Method for the producing alpha-1, 6-branched alpha-1, 4-glucans from sucrose

ABSTRACT

Nucleic acid molecules which encode a branching enzyme from a bacterium of the genus  Neisseria , vectors, host cell, plant cells and plants containing said nucleic acid molecules as well as starch obtainable from the plants described are described. Furthermore, an in-vitro method for producing α-1,6-branched α-1,4-glucans on the basis of sucrose and a combination of enzymes of an amylosucrase and a branching enzyme as well as the α-1,6-branched α-1,4-glucans obtainable by said method are described.

This application is a Divisional of co-pending U.S. application Ser. No.12/905,894 filed on Oct. 15, 2010, which is a Divisional of U.S.application Ser. No. 11/781,226 filed on Jul. 20, 2007 and now issued asU.S. Pat. No. 7,833,751, which is a Divisional of U.S. Ser. No.10/705,195 filed Nov. 10, 2003 and now issued as U.S. Pat. No.7,732,164, which is a Divisional of U.S. application Ser. No.09/807,063, filed on Jun. 11, 2001 and now issued as U.S. Pat. No.6,699,694 B1 which is a national phase under 35 U.S.C. §371 of PCTInternational Application No. PCT/EP99/07562 which has an Internationalfiling date of Oct. 8, 1999, which designated the United States and onwhich priority is claimed under 35 U.S.C. §120, the entire contents ofwhich are hereby incorporated by reference. This divisional applicationclaims priority under 35 U.S.C. §119 on Application No. 198 46 635.8filed in Germany on Oct. 9, 1998 and Application No. 199 24 342.5 filedin Germany on May 27, 1999, the entire contents of which are herebyincorporated by reference.

The present invention relates to nucleic acid molecules encoding abranching enzyme from bacteria of the genus Neisseria, vectors, hostcells, plant cells and plants containing such nucleic acid molecules aswell as starch obtainable from the plants described. Furthermore, thepresent invention relates to in-vitro methods for the production ofα-1,6-branched α-1,4-glucans on the basis of sucrose and a combinationof enzymes of an amylosucrase and a branching enzyme. Moreover, theinvention relates to glucans that are obtainable by the methoddescribed.

In many respects, α-1,6-branched α-1,4-glucans are of enormous interestsince they are suitable, for instance, as regards the production ofproducts in the pharmaceutical and cosmetic industry. They can be used,e.g. as binding agent for tablets, as carrier substances forpharmaceutical agents, as packaging material, as carrier substance forpowder additives, as UV-absorbing additive in sun creme and as carriersubstance of flavourings and scents.

In plants, α-1,6-branched α-1,4-glucans can mainly be found asamylopectin, a component of starch. In animals and in bacteria, glucansmainly occur in form of glycogen.

The polysaccharide starch is formed of chemically uniform basic buildingblocks, i.e. the glucose molecules, it is, however, a complex mixture ofdifferent forms of molecules which differ with regard to the degree ofpolymerization and branching and which, thus, differ strongly in theirphysico-chemical properties. It has to be differentiated between amylosestarch, which is an essentially non-branched polymer ofα-1,4-glycosidically linked glucose units, and the amylopectin starch,which is a branched polymer in which the branchings are formed due tothe presence of additional α-1,6-glycosidical linkings. According totextbooks (Voet and Voet, Biochemistry, John Wiley & Sons, 1990), theα-1,6-branchings occur after every 24 to 30 glucose residues on average,which corresponds to a branching degree of approximately 3% to 4%. Theindications as to the branching degree vary and depend on the origin ofthe respective starch (e.g. plant species, plant variety). In plantsthat are typically used for the industrial production of starch theshare of amylose in the overall share of starch varies between 10% and25%. Various approaches for the production of α-1,6-branchedα-1,4-glucans with different branching degrees have already beendescribed, with these approaches comprising the use of (transgenic)plants.

The heterologous expression of a bacterial glycogen synthase in potatoplants, for instance, leads to a slight decrease of the amylose content,to an increase in the branching degree and to a modification of thebranching pattern of the amylopectin when compared to wild type plants(Shewmaker et al., Plant. Physiol. 104 (1994), 1159-1166). Furthermore,it was observed that the heterologous expression of the branching enzymefrom E. coli (glgB) in amylose-free potato mutants (amf) (Jacobsen etal., Euphytica 44 (1989), 43-48) leads to amylopectin molecules whichhave 25% more branching points (Kortstee et al., Plant J. 10 (1996),83-90) than the control (amf). For isolating the glucans with differentbranching degrees, which were produced in transgenic plants, it isnecessary to carry out additional purification steps in order to remove,for example, the amylose component. These purification steps arelaborious and, therefore, time-consuming and cost-intensive.Furthermore, it is not possible to achieve a particular branching degreeby means of these approaches. What is more, due to varying experimentalconditions (environmental factors, location), such in-vivo methods varyconsiderably with regard to the quality of the product.

Glycogen has a higher branching degree than the amylopectin. Thispolysaccharide, too, contains α-1,6-branched α-1,4-glucans. Glycogenalso differs from starch in the average length of the side-chains and inthe degree of polymerization. According to textbooks (Voet and Voet,Biochemistry, John Wiley & Sons, 1990), glycogen contains, on average,an α-1,6-branching point after every 8 to 12 glucose residues. Thiscorresponds to a branching degree of approximately 8% to 12%. There arevarying indications as to the molecular weight of glycogen, which rangefrom 1 million to more than 1000 millions (D. J. Manners in: Advances inCarbohydrate Chemistry, Ed. M. L. Wolfrom, Academic Press, New York(1957), 261-298; Geddes et al., Carbohydr. Res. 261 (1994), 79-89).These indications, too, strongly depend on the respective organism oforigin, its state of nutrition and the kind of isolation of theglycogen. Glycogen is usually recovered from mussels (e.g. Mytillusedulis), from mammalian liver or muscles (e.g. rabbit, rat) (Bell etal., Biochem. J. 28 (1934), 882; Bueding and Orrell, J. Biol. Chem. 236(1961), 2854). This renders the production on an industrial scale verytime-consuming and cost-intensive.

The naturally-occurring α-1,6-branched α-1,4-glucans described, starchand glycogen, are very different depending on their content of1,6-glycosidic branchings. This holds true, amongst others, with regardto solubility, transparency, enzymatic hydrolysis, rheology, gelformation and retrogradation properties. For many industrialapplications, such variations in the properties, however, cannot alwaysbe tolerated.

In-vitro approaches are an alternative to the recovery of α-1,6-branchedα-1,4-glucans from plants or animal organisms. Compared to in-vivomethods, in-vitro methods are generally better to control and arereproducible to a greater extent since the reaction conditions in vitrocan be exactly adjusted in comparison with the conditions in a livingorganism. This usually allows the production of invariable products witha high degree of uniformity and purity and, thus, of high quality, whichis very important for any further industrial application. Thepreparation of products of a steady quality leads to a reduction ofcosts since the procedural parameter that are necessary for thepreparation do not have to be optimised for every preparation set-up.Another advantage of certain in-vitro methods is the fact that theproducts are free of the organisms used in the in-vivo method. This isabsolutely necessary for particular applications in the food andpharmaceutical industries.

In general, in-vitro methods can be divided into two different groups.

In the first group of methods, various substrates, such as amylose,amylopectin and glycogen, are subjected to the activity of a branchingenzyme.

Borovsky et al. (Eur. J. Biochem. 59 (1975), 615-625) were able to provethat using the branching enzyme from potato in connection with thesubstrate amylose leads to products that are similar to amylopectin, butthat differ from it in their structure.

Boyer and Preiss (Biochemistry 16 (1977), 3693-3699) showed, inaddition, that a purified branching enzyme (α-1,4-glucan: α-1,4-glucan6-glycosyltransferase) from E. coli may be used to increase thebranching degree of amylose or amylopectin.

If, however, glycogen from E. coli or rabbit liver is incubated with thebranching enzyme from E. coli, only a slight increase in the branchingdegree can be achieved (Boyer and Preiss, loc. cit.).

Rumbak et al. (J. Bacteriol. 173 (1991), 6732-6741), too, couldsubsequently increase the branching degree of amylose, amylopectin andglycogen by incubating these substrates with the branching enzyme fromButyrivibrio fibrisolvens.

Okada et al. made a similar approach (U.S. Pat. No. 4,454,161) toimprove the properties of starch-containing foodstuffs. They incubatedsubstances, such as amylose, amylopectin, starch or dextrin with abranching enzyme. This had advantageous effects on the durability offoodstuffs containing substances that were modified correspondingly.Furthermore, the patent application EP-A1 0 690 170 describes thereaction of jellied starch in an aqueous solution using a branchingenzyme. This results in starches having advantageous properties in theproduction of paper.

However, the aforementioned in-vitro methods have the disadvantage thatthey, due to the varying branching degree of the educts (e.g. starch,amylopectin, etc.), make it impossible to produce uniform products. Inaddition, it is not possible to intentionally control the branchingdegree and, what is more, the substrates used are quite expensive.

The other group of in-vitro methods comprises the de-novo synthesis ofα-1,6-branched α-1,4-glucans starting from various substrates(glucose-1-phosphate, ADP glucose, UDP glucose) using a combination ofenzymes that consists of a 1,4-glucan-chain-forming enzyme(phosphorylase, starch synthase, glycogen synthase) and a branchingenzyme.

Illingwort et al. (Proc. Nat. Acad. Sci. USA 47 (1961), 469-478) wereable to show for an in-vitro method using a phosphorylase A from muscles(organism unknown) in combination with a branching enzyme (organismunknown) that the de-novo synthesis of molecules similar to glycogenusing the substrate glucose-1-phosphate was possible. Boyer and Preiss(loc. cit.) combined the enzymatic activity of a phosphorylase fromrabbit muscles or a glycogen synthase from E. coli with the activity ofa branching enzyme from E. coli using the substrate glucose-1-phosphateor UDP glucose and in this way generated branched α-glucans. Borovsky etal. (Eur. J. Biochem. 59 (1975), 615-625), too, analysed the de-novosynthesis of α-1,6-branched α-1,4-glucans from glucose-1-phosphate usinga branching enzyme from potato in combination with a phosphorylase(1,4-α-D-glucan: orthophosphate α-glycosyltransferase [EC 2.4.1.1]) frommaize. Doi (Biochimica et Biophysica Acta 184 (1969), 477-485) showedthat the enzyme combination of a starch synthase (ADP-D-glucose:α-1,4-glucan α-4-glucosyltransferase) from spinach and a branchingenzyme from potato using the substrate ADP glucose resulted in productssimilar to amylopectin. Parodi et al. (Arch. Biochem. Biophys. 132(1969), 11-117) used a glycogen synthase from rat liver combined with abranching enzyme from rat liver for the de-novo synthesis of branchedglucans from UDP glucose. They obtained a polymer which was similar tonative glycogen and which differs from the polymers that are based onglucose-1-phosphate.

This second group of in-vitro methods, too, has the disadvantage thatthe substrates, e.g. glucose-1-phosphate, UDP glucose and ADP glucose,are very expensive. Furthermore, it does not seem to be possible eitherto intentionally control the branching degree.

Büttcher et al. (J. Bacteriol. 179 (1997), 3324-3330) describe anin-vitro method for the production of water-insoluble α-1,4-glucansusing an amylosucrase and sucrose as substrates. However, only linearα-1,4-glucans without branchings are synthesized.

Thus, the technical problem underlying the present invention is toprovide a method allowing the cheap production of α-1,6-branchedα-1,4-glucans for industrial purposes, as well as nucleic acid moleculesencoding the enzymes that may be used in said methods, in particularbranching enzymes.

This technical problem has been solved by providing the embodimentscharacterised in the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 schematically shows the structure of the plasmid pBB48 (DSM12425).

FIG. 2 shows a number of α-1,4-glucans having a varying degree ofα-1,6-branchings which were produced by means of the method of theinvention and which were subsequently dyed with Lugol's solution.

From left to right: amylosucrase (left), amylosucrase+decreasing amountsof branching enzyme activity. The maximum absorption of thecorresponding samples were: 615 nm, 483 nm, 500 nm, 526 nm, 534 nm, 560nm, 577 nm.

FIG. 3 shows a HPLC chromatograph of a highly branched process product(A) which has been debranched with isoamylase and a rat liver glycogensample (B) which has been debranched with isoamylase.

FIG. 4 shows the scheme of the methylation analysis.

FIG. 5 shows a diagram of the results of the analysis of sample 7described in Examples 9 and 10 after one and after two methylationsteps. The values for the 2,3,6-methylation are 96.12% and 96.36%,respectively.

FIG. 6 shows a graphic illustration of the shares in terminal (“2346Me”) and 6-linked (“23 Me”) glucose units of the glucan samplesexamined.

FIGS. 7 and 8 show gas chromatographs of the samples 3 and 7 describedin the Examples.

FIG. 9 schematically shows the plasmid pBE-fnr-Km.

FIG. 10 shows an activity gel for the branching enzyme.

FIG. 11 shows the schematic illustration of an RVA profile.

FIG. 12 shows the distribution of granule size of the lines 143-13A and143-59A compared to the wild type.

FIG. 13 shows the microscopic magnification of the starch granules ofthe lines 143-13A, 143-34A and 143-59A in comparison with the starchgranules of wild type plants (light microscope by Leitz, Germany).

FIG. 14 shows the gel texture of the starches of different transgeniclines compared to starches from wild type plants. The texture wasdetermined by means of a texture analyzer.

FIG. 15 shows the RVA profile of the starches of the lines 143-11A,143-13A, 143-59A compared to the wild type.

FIGS. 16 to 18 show the results of HPLC chromatographies which representthe pattern of the distribution of the side-chains of the lines 143-WT(=wild type), 143-13A and 143-59A.

FIG. 19 shows the elution gradient that was used for thechromatographies depicted in FIGS. 16 to 18.

FIG. 20 shows the percentage deviation of side-chains having certainchain lengths of the starches analysed in FIGS. 16 to 18 from the wildtype.

Therefore, the present invention relates to nucleic acid moleculesencoding a branching enzyme (EC 2.4.1.18) from bacteria of the genusNeisseria selected from the group consisting of

-   (a) nucleic acid molecules encoding a protein which comprises the    amino acid sequence depicted in SEQ ID NO. 2;-   (b) nucleic acid molecules comprising the nucleotide sequence of the    coding region which is depicted in SEQ ID NO. 1;-   (c) nucleic acid molecules encoding a protein which comprises the    amino acid sequence that is encoded by the insert of the plasmid DSM    12425;-   (d) nucleic acid molecules comprising the region of the insert of    the plasmid DSM 12425, which encodes a branching enzyme from    Neisseria denitrificans;-   (e) nucleic acid molecules encoding a protein the sequence of which    has within the first 100 amino acids a homology of at least 65% with    regard to the sequence depicted in SEQ ID NO. 2;-   (f) nucleic acid molecules the complementary strand of which    hybridizes to a nucleic acid molecule according to (a), (b),    (c), (d) and/or (e) and which encode a branching enzyme from a    bacterium of the genus Neisseria; and-   (g) nucleic acid molecules the nucleic acid sequence of which    differs from the sequence of a nucleic acid molecule according    to (f) due to the degeneracy of the genetic code.

The nucleic acid sequence depicted in SEQ ID NO. 1 is a genomic sequencewhich comprises a coding region for a branching enzyme from Neisseriadenitrificans. A plasmid containing said DNA sequence has been depositedas DSM 12425. By means of said sequence or said molecule, the personskilled in the art can now isolate homologous sequences from otherNeisseria species or Neisseria strains. He/she may do so usingconventional methods, like screening of cDNA or genomic libraries withsuitable hybridization probes. The homologous sequences may also beisolated as described in Example 1. Thus, it is possible, for example,to identify and isolate nucleic acid molecules that hybridize to thesequence depicted in SEQ ID NO. 1 and that encode a branching enzyme.

The nucleic acid molecules of the invention may, in principle, encode abranching enzyme from any bacterium of the genus Neisseria, theypreferably encode a branching enzyme from Neisseria denitrificans.

According to the present invention, the term “hybridization” meanshybridization under conventional hybridization conditions, preferablyunder stringent conditions as have been described, e.g. in Sambrook etal., Molecular Cloning, A Laboratory Manual, 2^(nd) edition (1989) ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y. The term“hybridization” is particularly preferred to mean a hybridization underthe following conditions:

-   hybridization buffer: 2×SSC; 10×Denhardt solution (Fikoll    400+PEG+BSA; at a ratio of 1:1:1); 0.1% SDS; 5 mM EDTA; 50 mM    Na₂HPO₄; 250 μg/ml herring sperm DNA; 50 μg/ml tRNA; or    -   25 M sodium phosphate buffer, pH 7.2; 1 mM EDTA; 7% SDS-   hybridization temperature: T=65 to 68° C.-   washing buffer: 0.2×SSC; 0.1% SDS-   washing temperature: T=65 to 68° C.

Nucleic acid molecules hybridizing to the nucleic acid molecules of theinvention may, in principle, be derived from any bacterium of the genusNeisseria which expresses a corresponding protein, preferably they arederived from Neisseria denitrificans. Nucleic acid molecules hybridizingto the molecules of the invention, may, for instance, be isolated fromgenomic or from cDNA libraries. Such nucleic acid molecules can beidentified and isolated using the nucleic acid molecules of theinvention or parts of said molecules or the reverse complements of saidmolecules, e.g. by hybridizing according to standard techniques (cf.Sambrook et al., Molecular Cloning, A Laboratory Manual, 2^(nd) edition(1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.) orby amplification by means of PCR.

As hybridization probe nucleic acid molecules can be used which haveexactly or essentially the nucleotide sequence depicted in SEQ ID NO. 1or parts thereof. The fragments used as hybridization probes may also besynthetic fragments which have been produced by means of conventionalsynthesis techniques and the sequence of which is essentially identicalto the one of a nucleic acid molecule of the invention. If genes havebeen identified and isolated to which the nucleic acid sequences of theinvention hybridize, the sequence should be determined and theproperties of the proteins encoded by said sequence should be analysedto find out whether they are branching enzymes. For this purpose, it isparticularly suitable to compare the homology on the nucleic acid andamino acid sequence level and to determine the enzymatic activity.

The molecules hybridizing to the nucleic acid molecules of the inventioncomprise, in particular, fragments, derivatives and allelic variants ofthe above-described nucleic acid molecules encoding a branching enzymefrom bacteria of the genus Neisseria, preferably from Neisseriadenitirificans. In this context, the term “derivative” means that thesequences of said molecules differ from the sequences of theaforementioned nucleic acid molecules in one of more positions and havea high degree of homology to said sequences. Homology, in this context,means that there is, over the entire length, a sequence identity of atleast 60%, in particular an identity of at least 70%, preferably of morethan 80%, more preferably of more than 90% and most preferably of atleast 95%. The deviations from the above-described nucleic acidmolecules may be caused by, e.g. deletion, addition, substitution,insertion or recombination.

Furthermore, homology means that there is a functional and/or structuralequivalence between the respective nucleic acid molecules or theproteins encoded by these. The nucleic acid molecules which arehomologous to the aforementioned molecules and which are derivatives ofsaid molecules are usually variations of said molecules which aremodifications that have the same biological functions. These may be bothnaturally-occurring variations, e.g. sequences from other Neisseriaspecies or Neisseria strains and mutations with these mutationsoccurring naturally or being introduced by directed mutagenesis.Furthermore, the variations may be sequences produced synthetically. Theallelic variants may be both naturally-occurring variants and variantsthat have been produced synthetically or by recombinant DNA techniques.

The proteins encoded by the different variants of the nucleic acidmolecules of the invention have certain characteristics in common. Thesemay include, for instance, biological activity, molecular weight,immunological reactivity, conformation, etc., as well as physicalproperties, such as the migration behaviour in gel electrophoreses,chromatographic behaviour, sedimentation coefficients, solubility,spectroscopic properties, stability; pH optimum, temperature optimum,etc.

The molecular weight of the branching enzyme from Neisseriadenitrificans is 86.3 kDa, with the molecular weight being deduced fromthe amino acid sequence. Hence, the deduced molecular weight of aprotein of the invention preferably ranges from 70 kDa to 100 kDa, morepreferably from 77 kDa to 95 kDa and most preferably it is about 86 kDa.

The present invention also relates to nucleic acid molecules encoding aprotein having the enzymatic activity of a branching enzyme with theencoding protein having a homology of at least 65%, preferably of atleast 80% and most preferably of at least 95% in the region of theN-terminus, preferably in the first 100 amino acids, more preferably inthe first 110 amino acids and most preferably in the first 120 aminoacids to the amino acid sequence depicted in SEQ ID NO. 2.

In another embodiment, the present application relates to nucleic acidmolecules encoding a protein having activity of a branching enzyme, theprotein comprising at least one, preferably at least 5, more preferablyat least 10 and most preferably at least 20 of the following peptidemotifs:

(a) MNRNRHI, (SEQ ID NO. 8) (b) RPDAHH, (SEQ ID NO. 9) (c) HAPDYAL,(SEQ ID NO. 10) (d) EGEAA, (SEQ ID NO. 11) (e) DDYRF, (SEQ ID NO. 12)(f) SALQH, (SEQ ID NO. 13) (g) YETLG, (SEQ ID NO. 14) (h) VSGVR,(SEQ ID NO. 15) (i) VSVIG, (SEQ ID NO. 16) (j) FNGWD, (SEQ ID NO. 17)(k) LYKFS, (SEQ ID NO. 18) (l) PYAFG, (SEQ ID NO. 19) (m) RPTTAS,(SEQ ID NO. 20) (n) FRRRA, (SEQ ID NO. 21) (o) DELVNY, (SEQ ID NO. 22)(p) LPLSEY, (SEQ ID NO. 23) (q) YQATGL, (SEQ ID NO. 24) (r) DDHGL,(SEQ ID NO. 25) (s) HQDWN, (SEQ ID NO. 26) (t) DGIRV, (SEQ ID NO. 27)(u) YGGSEN, (SEQ ID NO. 28) (v) SFAEES, (SEQ ID NO. 29) (w) DPVHR,(SEQ ID NO. 30) (x) WQQFAN, (SEQ ID NO. 31) (y) EILNS, (SEQ ID NO. 32)(z) ATEIQTAL, (SEQ ID NO. 33) (aa) VKDKQAKAK. (SEQ ID NO. 34)

The nucleic acid molecules of the invention may be any nucleic acidmolecules, in particular DNA or RNA molecules, e.g. cDNA, genomic DNA,mRNA, etc. They may be naturally-occurring molecules or moleculesproduced by means of genetic or chemical synthesis techniques. They maybe single-stranded molecules which either contain the coding or thenon-coding strand, or they may also be double-stranded molecules.

Furthermore, the present invention relates to nucleic acid moleculeswhich are at least 15, preferably more than 50 and most preferably morethan 200 nucleotides in length, these nucleic acid moleculesspecifically hybridizing to at least one nucleic acid molecule of theinvention. In this context, the term “specifically hybridizing” meansthat said molecules hybridize to nucleic acid molecules encoding aprotein of the invention, however, not to nucleic acid moleculesencoding other proteins. The term “hybridizing” means preferablyhybridizing under stringent conditions (see above). In particular, theinvention relates to nucleic acid molecules which hybridize totranscripts of nucleic acid molecules of the invention and which, thus,can prevent the translation thereof. Such nucleic acid molecules whichspecifically hybridize to the nucleic acid molecules of the inventionmay, for instance, be components of anti-sense constructs or ribozymesor may be used as primers for amplification by means of PCR.

Moreover, the invention relates to vectors, in particular plasmids,cosmids, viruses, bacteriophages and other vectors that are usually usedin genetic engineering and that contain the above-described nucleic acidmolecules of the invention.

In a preferred embodiment, the nucleic acid molecules contained in thevectors are linked in sense-orientation to regulatory elementsguaranteeing expression in prokaryotic or eukaryotic cells. In thiscontext, the term “expression” means both transcription or transcriptionand translation.

The expression of the nucleic acid molecules of the invention inprokaryotic cells, e.g. in Escherichia coli, allows, for instance, amore exact characterisation of the enzymatic activities of the proteinsencoded. In addition, it is possible to introduce various mutations intothe nucleic acid molecules of the invention by means of conventionaltechniques of molecular biology (cf. e.g. Sambrook et al., loc. cit.).This leads to the synthesis of proteins the properties of which haveoptionally been modified. It is also possible to produce deletionmutants by continued deletion of the 5′ or 3′ end of the encoding DNAsequence, which results in the generation of nucleic acid moleculesleading to the synthesis of correspondingly shortened proteins.Moreover, it is possible to introduce point mutations at positions thatinfluence, for instance, the enzyme activity or the regulation of theenzyme. In this way, mutants may be generated that have a modified Kmvalue or that are no longer subjected to the usual regulation mechanismsin the cells via allosteric regulation or covalent modification. Inaddition, mutants may be produced which have a modified substrate orproduct specificity. Furthermore, mutants may be produced which have amodified activity-temperature profile. The genetic manipulation inprokaryotic cells may be carried out according to methods known to theskilled person (cf. Sambrook et al., loc. cit.).

Regulatory sequences for the expression in prokaryotic organisms, e.g.E. coli, and in eukaryotic organisms have been sufficiently described inthe literature, in particular sequences for the expression in yeast,such as Saccharomyces cerevisiae. Methods in Enzymology 153 (1987),383-516 and Bitter et al. (Methods in Enzymology 153 (1987), 516-544)give an overview of various systems for the expression for proteins invarious host organisms.

Preferably, the nucleic acid molecule of the invention which has beeninserted in a vector of the invention is modified in such a way that itis easier to isolate the encoded protein from the culture medium afterit had been expressed in a suitable host organism. There is, forinstance, the possibility of expressing the encoded branching enzyme asa fusion protein together with a further polypeptide sequence thespecific binding properties of which allow the isolation of the fusionprotein by means of affinity chromatography (cf. Chong et al., Gene 192(1997), 271-281; Hopp et al., Bio/Technology 6 (1988), 1204-1210;Sassenfeld, Trends Biotechnol. 8 (1990), 88-93).

Furthermore, the nucleic acid molecule contained in vector of theinvention is preferred to comprise nucleotide sequences which allow thesecretion of the branching enzyme into the culture medium. Preferably, asequence is used which codes for the signal peptide of the α-CGTase fromKlebsiella oxytoca M5A1 (Fiedler et al., J. Mol. Biol. 256 (1996),279-291; Genebank acc. no. X86014, CDS 11529-11618). The recovery andthe purification is made easier by the secretion of the enzyme into theculture medium. A disruption of the cells is avoided and the enzyme canbe recovered from the culture medium with conventional methods, such asdialysis, osmosis, chromatographic methods, etc. being used for removingresiduary components of the culture medium.

Furthermore, the vectors of the invention may comprise other functionalunits which may bring about a stabilisation of the vector in a hostorganism, such as a bacterial replication origin or the 2μ-DNA for thestabilisation in S. cerevisiae.

In another embodiment, the invention relates to host cells, inparticular to prokaryotic or eukaryotic cells which have beentransformed with a nucleic acid molecule or a vector as described above,as well as to cells which are derived from said host cells and whichcontain the described nucleic acid molecules or vectors. The host cellsmay be bacterial cells (e.g. E. coli) or fungal cells (e.g. yeast, inparticular S. cerevisiae), as well as plant or animal cells. The term“transformed” means that the cells of the invention have beengenetically modified with a nucleic acid molecule of the invention in sofar as they contain at least one nucleic acid molecule of the inventionin addition to their natural genome. Said nucleic acid molecule may bepresent free in the cell, optionally as self-replicating molecule, or itmay be stably integrated into the genome of the host cell.

The host cells are preferred to be microorganisms. Within the presentinvention, such microorganisms may be all bacteria and all protista(e.g. fungi, in particular yeasts and algae) as have been defined, forinstance, in Schlegel “Allgemeine Mikrobiologie” (Georg Thieme Verlag(1985), 1-2).

The host cells of the invention are particularly preferred to be plantcells. In principle, these may include plant cells from any plantspecies, i.e. both from monocotyledonous and dicotyledonous plants.Preferably, said cells are plant cells from agricultural useful plants,i.e. plants that people cultivate for nutritional or technical purposes,in particular, for industrial purposes. The invention preferably relatesto plants cells from fibre-forming plants (e.g. flax, hemp, cotton),oil-storing plants (e.g. rape, sunflower, soy bean), sugar-storingplants (e.g. sugar beat, sugar cane, sugar millet, banana) andprotein-storing plants (e.g. leguminoses).

In another embodiment, the invention relates to plant cells from forageplants (e.g. forage grass and pasture grass (alfalfa, clover, etc.)),vegetable plants (e.g. tomato, lettuce, chicory).

In a preferred embodiment, the invention relates to plant cells fromstarch-storing plants (e.g. wheat, barley, oat, rye, potato, maize,rice, pea, cassava, mung bean). Plant cells from maize, rice, wheat andpotato plants are particularly preferred.

Moreover, the present invention relates to a method for producing abranching enzyme from bacteria of the genus Neisseria. In said method,the host cells of the invention are cultivated under conditions allowingthe protein to be expressed and the protein is recovered from theculture, i.e. from the cells and/or the culture medium. Preferably, ahost organism that secretes the branching enzyme is used.

Furthermore, the present invention relates to a method for producing abranching enzyme from bacteria of the genus Neisseria with the proteinbeing produced in an in-vitro transcription and translation system usinga nucleic acid molecule of the invention. The person skilled in the artis familiar with such systems.

The invention also relates to proteins which are encoded by the nucleicacid molecules of the invention or which are obtainable by a method ofthe invention.

Furthermore, the present invention relates to antibodies whichspecifically recognise a protein of the invention. These antibodies maybe, for instance, monoclonal or polyclonal antibodies. They may also befragments of antibodies which recognise the proteins of the invention.The person skilled in the art is familiar with methods for producingsaid antibodies or fragments.

Furthermore, the present invention relates to the use of a branchingenzyme of the invention for the production of α-1,6-branchedα-1,4-glucans in in-vitro systems.

In particular, the present invention also relates to transgenic plantcells which contain the nucleic acid molecules or vectors of theinvention. Preferably, the cells of the invention are characterised inthat the nucleic acid molecule of the invention which has beenintroduced is stably integrated into the genome and is controlled by apromoter active in plant cells.

There is a plurality of promoters or regulatory elements at disposal forexpressing a nucleic acid molecule of the invention in plant cells. Inprinciple, all promoters, enhancers, terminators, etc. that are activein plants are regulatory elements for the expression in plant cells.Basically any promoter which is functional in the plants selected forthe transformation can be used. With regard to the plant species used,the promoter can be homologous or heterologous. Said promoter may beselected in such a way that the expression takes place in a constitutivemanner or only in a particular tissue, at a certain time in thedevelopment of the plant or at a time that is determined by externalinfluence. Examples of suitable promoters are the 35S promoter of thecauliflower mosaic virus (Odell et al., Nature 313 (1985), 810-812 orU.S. Pat. No. 5,352,605), which ensures a constitutive expression in alltissues of a plant, and the promoter construct described in WO/9401571.The ubiquitin promoter (cf. e.g. U.S. Pat. No. 5,614,399) and thepromoters of the polyubiquitin genes from maize (Christensen et al.,loc. cit.) are further examples. However, also promoters which are onlyactivated at a time determined by external influence (cf. e.g.WO/9307279) can be used. Promoters of heat shock proteins allowing asimple induction may be of particular interest. Furthermore, promoterscan be used which lead to the expression of downstream sequences in acertain tissue of the plant, e.g. in photosynthetically active tissue.Examples thereof are the ST-LS1 promoter (Stockhaus et al., Proc. Natl.Acad. Sci. USA 84 (1987), 7943-7947; Stockhaus et al., EMBO J. 8 (1989),2445-2451), the Ca/b promoter (cf. e.g. U.S. Pat. No. 5,656,496, U.S.Pat. No. 5,639,952, Bansal et al., Proc. Natl. Acad. Sci. USA 89 (1992),3654-3658) and the Rubisco SSU promoter (cf. e.g. U.S. Pat. No.5,034,322 and U.S. Pat. No. 4,962,028). In addition, promoters that areactive in the starch-storing organs of plants to be transformed are tobe mentioned. It is, for instance, the maize kernels in maize, whereasin potatoes, it is the tubers. For over-expressing the nucleic acidmolecules of the invention in potato, the tuber-specific patatin genepromoter B33 (Rocha-Sosa et al., EMBO J. 8 (1989), 23-29) can, forexample, be used. Seed-specific promoters have already been describedfor various plant species. The USP promoter from Vicia faba, whichguarantees a seed-specific expression in V. faba and other plants(Fiedler et al., Plant Mol. Biol. 22 (1993), 669-679; Bäumlein et al.,Mol. Gen. Genet. 225 (1991), 459-467) is an example thereof.

Moreover, fruit-specific promoters as described in WO 91/01373 can alsobe used. Promoters for an endosperm-specific expression, such as theglutelin promoter (Leisy et al., Plant Mol. Biol. 14 (1990), 41-50;Zheng et al., Plant J. 4 (1993), 357-366), the HMG promoter from wheat,the USP promoter, the phaseolin promoter or promoters of zein genes frommaize (Pedersen et al., Cell 29 (1982), 1015-1026; Quatroccio et al.,Plant Mol. Biol. 15 (1990), 81-93) are particularly preferred. By meansof endosperm-specific promoters it is possible to increase the amountsof transcripts of the nucleic acid molecules of the invention in theendosperm in comparison with the endosperm of corresponding wild typeplants.

The shrunken-1-promoter (sh-1) from maize (Werr et al., EMBO J. 4(1985), 1373-1380) is particularly preferred.

In addition, there may be a terminator sequence which is responsible forthe correct termination of the transcription and the addition of apoly-A tail to the transcript having the function of stabilising thetranscripts. Such elements have been described in the literature (cf.e.g. Gielen et al., EMBO J. 8 (1989), 23-29) and may be exchanged atwill.

Therefore, it is possible to express the nucleic acid molecules of theinvention in plant cells.

Thus, the present invention also relates to a method for producingtransgenic plant cells comprising introducing a nucleic acid molecule ora vector of the invention into plant cells. The person skilled in theart has various plant transformation systems at disposal, e.g. the useof T-DNA for transforming plant cells has been examined extensively andhas been described in EP-A-120 516; Hoekema: The Binary Plant VectorSystem, Offsetdrukkerij Kanters B. V., Alblasserdam (1985), Chapter V,Fraley, Crit. Rev. Plant. Sci., 4, 1-46 and An, EMBO J. 4 (1985),277-287.

For transferring the DNA in the plant cells, plant explants may suitablybe co-cultivated with Agrobacterium tumefaciens or Agrobacteriumrhizogenes. Whole plants may then be regenerated from the infected plantmaterial (e.g. parts of leaves, stem segments, roots and protoplasts orplant cells cultivated in suspensions) in a suitable medium which cancontain antibiotics or biocides for selecting transformed cells. Theplants obtained in that way can then be examined for the presence of theDNA introduced. Other possibilities of introducing foreign DNA using thebiolistic method or by protoplast transformation are known (cf.Willmitzer, L. 1993 Transgenic plants. In: Biotechnology, A Multi-VolumeComprehensive Treatise (H. J. Rehm, G. Reed, A. Pühler, P. Stadler,eds.), Vol. 2, 627-659, VCH Weinheim-New York-Basel-Cambridge).

Alternative systems for transforming monocotyledonous plants are thetransformation by means of the biolistic method, the electrically orchemically induced DNA absorption in protoplasts, the electroporation ofpartially permeabilised cells, the microinjection of DNA in theinflorescence, the microinjection of DNA in microspores and pro-embryos,the DNA absorption through germinating pollens and the DNA absorption inembryos by swelling (cf. e.g. Lusardi, Plant J. 5 (1994), 571-582;Paszowski, Biotechnology 24 (1992), 387-392).

While the transformation of dicotyledonous plants via Ti-plasmid vectorsystems by means of Agrobacterium tumefaciens is well established, morerecent studies point to the fact that monocotyledonous plants, too, canindeed be transformed by means of vectors based on Agrobacterium (Chan,Plant Mol. Biol. 22 (1993), 491-506; Hiei, Plant J. 6 (1994), 271-282;Bytebier, Proc. Natl. Acad. Sci. USA 84 (1987), 5345-5349; Raineri,Bio/Technology 8 (1990), 33-38; Gould, Plant Physiol. 95 (1991),426-434; Mooney, Plant, Cell Tiss. & Org. Cult. 25 (1991), 209-218; Li,Plant Mol. Biol. 20 (1992), 1037-1048).

In the past, three of the above transformation systems could beestablished for various cereals: the electroporation of tissue, thetransformation of protoplasts and the DNA transfer by particlebombardment in regenerable tissue and cells (for an overview see Jane,Euphytica 85 (1995), 35-44). The transformation of wheat has beendescribed several times in the literature (for an overview seeMaheshwari, Critical Reviews in Plant Science 14 (2) (1995), 149-178).

In particular, the transformation of maize has been described severaltimes in the literature (cf. e.g. WO 95/06128, EP 0513849, EO 0465875,EP 292435; Fromm et al., Biotechnology 8 (1990), 833-844; Gordon-Kamm etal., Plant Cell 2 (1990), 603-618; Koziel et al., Biotechnology 11(1993), 194-200; Moroc et al., Theor. Appl. Genet. 80 (1990), 721-726).

The successful transformation of other kinds of cereals has also beendescribed, e.g. for barley (Wan and Lemaux, loc. cit.; Ritala et al.,loc. cit.; Krens et al., Nature 296 (1982), 72-74) and for wheat (Nehraet al., Plant J. 5 (1994), 285-297).

For expressing the nucleic acid molecules of the invention in plants itis, in principle, possible for the synthesized protein to be located inany compartment of the plant cell. The coding region must optionally belinked to DNA sequences which guarantee the localisation in therespective compartment in order to achieve localisation in a particularcompartment. Such sequences are known (cf. e.g. Braun, EMBO J. 11(1992), 3219-3227; Sonnewald, Plant J. 1 (1991), 95-106; Rocha-Sosa,EMBO J. 8 (1989), 23-29).

As plastidial signal sequence, for instance, the one offerrodoxin:NADP+oxidoreductase (FNR) from spinach can be used. Saidsequence contains the 5′ non-translated region and the flanking transitpeptide sequence of the cDNA of the plastidial proteinferrodoxin:NADP+oxidoreductase from spinach (nucleotide −171 to +165;Jansen et al., Current Genetics 13 (1988), 517-522).

Furthermore, the transit peptide of the waxy protein from maize plus thefirst 34 amino acids of the mature waxy protein (Klösgen et al., Mol.Gen. Genet. 217 (1989), 155-161) may also be used as plastidial signalsequence. In addition, the transit peptide of the waxy protein frommaize (cf. above) may also be used without the 34 amino acids of themature waxy protein.

Moreover, it is also thinkable to use to following plastidial signalsequences: the signal sequence of the ribulose biphosphate carboxylasesmall subunit (Wolter et al., Proc. Natl. Acad. Sci. USA 85 (1988),846-850; Nawrath et al., Proc. Natl. Acad. Sci. USA 91 (1994),12760-12764); the signal sequence of the NADP malate dehydrogenase(Gallardo et al., Planta 197 (1995), 324-332); the signal sequence ofthe glutathione reductase (Creissen et al., Plant J. 8 (1995), 167-175).

Therefore, the present invention also relates to transgenic plant cellsthat were transformed with one or more of the nucleic acid molecule(s)of the invention, as well as to transgenic plant cells that are derivedfrom cells transformed in such a way. Such cells contain one or morenucleic acid molecule(s) of the invention with said molecule(s)preferably being linked to regulatory DNA elements which guarantee thetranscription in plant cells, in particular with a promoter. Such cellscan be differentiated from naturally-occurring plant cells in that theycontain at least one nucleic acid molecule of the invention.

The transgenic plant cells may be regenerated to whole plants usingtechniques well-known to the person skilled in the art. The plantsobtainable by means of regeneration of the transgenic plant cells of theinvention are also a subject matter of the present invention.

Moreover, plants containing the aforementioned plant cells are a subjectmatter of the present invention. The plants of the invention may, inprinciple, be plants of any plant species, i.e. both monocotyledonousand dicotyledonous plants. They are preferred to be useful plants, i.e.plants which are cultivated for nutritional or technical, in particular,industrial purposes. Preferably, the invention relates to plant cellsfrom fibre-forming plants (e.g. flax, hemp, cotton), oil-storing plants(e.g. rape, sunflower, soy bean), sugar-storing plants (e.g. sugar beat,sugar cane, sugar millet, banana) and protein-storing plants (e.g.leguminoses).

In another embodiment, the invention relates to forage plants (e.g.forage grass and pasture grass (alfalfa, clover, etc.)), vegetableplants (e.g. tomato, lettuce, chicory).

In a preferred embodiment, the invention relates to starch-storingplants (e.g. wheat, barley, oat, rye, potato, maize, rice, pea, cassava,mung bean), plant cells from maize, rice, wheat and potato plants areparticularly preferred.

In a preferred embodiment, the cells of the plants of the invention havean increased activity of the protein of the invention in comparison withcorresponding plant cells of wild type plants that have not beengenetically modified. These are preferably cells of starch-storingtissue, in particular cells of tubers or of the endosperm, mostpreferably cells of potato tubers or the endosperm of maize, wheat orrice plants.

Within the meaning of the present invention, the term “increase of theactivity” means an increase in the expression of a nucleic acid moleculeof the invention which encodes a protein with branching enzyme activity,an increase in the amount of protein with branching enzyme activityand/or an increase in the activity of a protein with branching enzymeactivity in the plants.

The increase in the expression can, for instance, be determined bymeasuring the amount of transcripts coding for said proteins, e.g. bymeans of Northern blot analysis or RT-PCR. In this context, the term“increase” preferably means an increase in the amount of transcripts byat least 10%, preferably by at least 20%, more preferably by at least50% and most preferably by at least 75% in comparison with plant cellsthat have not been genetically modified.

The amount of proteins with branching enzyme activity may, for example,be determined by Western blot analysis. In this context, the term“increase” preferably means that the amount of proteins with branchingenzyme activity is increased by at least 10%, preferably by at least20%, more preferably by at least 50% and most preferably by at least 75%in comparison with corresponding cells that have not been geneticallymodified.

An increase in the activity of the branching enzyme can, for instance,be determined according to the method described in Lloyd et al.(Biochem. J. 338 (1999), 515-521). In this context, the term “increase”preferably means that the branching enzyme activity is increased by atleast 10%, preferably by at least 20%, more preferably by at least 50%and most preferably by at least 75%.

Surprisingly, it was found that plants containing plant cells of theinvention with an increased activity of a branching enzyme synthesize amodified starch compared to corresponding wild type plants that have notbeen genetically modified. The modified starch may, for instance, bemodified with regard to its physio-chemical properties, in particularthe amylose/amylopectin ratio, the branching degree, the average chainlength, the phosphate content, the viscosity, the size of the starchgranule, the distribution of the side-chains and/or the form of thestarch granule in comparison with starch synthesized in wild typeplants. As a consequence, this modified starch is more suitable forparticular purposes.

Furthermore, it was surprisingly found that in plant cells in which theactivity of the branching enzyme of the invention is increased, thecomposition of the starch is modified in such a way that it has a highergel texture and/or a reduced phosphate content and/or a reduced peakviscosity and/or a reduced pastification temperature and/or a reducedsize of the starch granule and/or a modified distribution of theside-chains in comparison with starch from corresponding wild typeplants.

In this context, the term “increased gel texture” means an increase byat least 10%, preferably by at least 50%, more preferably by at least100%, by at least 200% and most preferably by at least 300% incomparison with the gel texture of starch from wild type plants. The geltexture is determined as described below.

Within the meaning of the present invention, the term “reduced phosphatecontent” means that the overall content of covalently bound phosphateand/or the content of phosphate in the C-6 position of the starchsynthesized in the plant cells of the invention is reduced by at least20%, preferably by at least 40%, more preferably by at least 60% andmost preferably by at least 80% in comparison with the starch from plantcells of corresponding wild type plants.

The overall phosphate content or the content of phosphate in the C-6position may be determined according to the method as described below.

Within the meaning of the present invention, the term “reduced peakviscosity” means that the peak viscosity is reduced by at least 10%,preferably by at least 25%, more preferably by at least 50% and mostpreferably by at least 75% in comparison with the peak viscosity ofstarches from wild type plants.

Within the meaning of the present invention, the term “reducedpastification temperature” means that the pastification temperature isreduced by at least 0.5° C., preferably by at least 1.0° C., morepreferably by at least 2.0° C., most preferably by at least 3.0° C. incomparison with the pastification temperature of starches from wild typeplants.

The peak viscosity and the pastification temperature can be determinedwith a Rapid Visco Analyzer in the manner described below.

The skilled person is familiar with the terms “peak viscosity” and“pastification temperature”.

The term “reduced size of the starch granule” means that the percentageproportion of the starch granules having a size of up to 15 μm isincreased by at least 10%, preferably by at least 30%, more preferablyby at least 50%, 100% and most preferably by at least 150% in comparisonwith wild type plants.

The size of the starch granules is determined by means of aphotosedimentometer of the type “Lumosed” by Retsch, GmbH, Germany inthe manner described below.

In this context, the term “modified distribution of the side-chains”means that the proportion of side-chains with a DP of 6 to 9 isincreased by at least 25%, preferably by at least 50%, more preferablyby at least 100% and most preferably by at least 200% in comparison withthe proportion of side-chains with a DP of 6 to 9 of amylopectin fromwild type plants.

In another embodiment of the invention, the term a “modifieddistribution of side-chains” means that the proportion of side-chainswith a DP of 6 to 8, preferably of 6 to 7 is increased by at least 25%,preferably by at least 50%, more preferably by at least 100% and mostpreferably by at least 200% in comparison to the proportion ofside-chains with the corresponding degree of polymerization ofamylopectin from wild type plants.

The proportion of side chains is established by determining thepercentage proportion of a particular side-chain with regard to theoverall share of all side-chains. The overall share of all side-chainsis established by determining the overall area below the peaks whichrepresent the polymerization degrees of DP 6 to 30 in the HPLCchromatograph. The percentage proportion of a particular side-chain withregard to the overall share of all side-chains is established bydetermining the ratio of the area below the peak that represents saidside-chain in the HPLC chromatograph to the overall area. Preferably,the program AI-450, version 3.31 by Dionex, USA, is used.

In another embodiment, the present invention relates to a starch theamylopectin of which has side-chains with a DP of 5 compared to theamylopectin of starches of wild type plants.

Furthermore, the present invention relates to a method for producing atransgenic plant which synthesizes a modified starch, wherein

-   (a) a plant cell is genetically modified by introducing a nucleic    acid molecule of the invention and/or a vector of the invention the    presence or expression of which leads to an increase in the activity    of a protein having the activity of a branching enzyme;-   (b) a plant is regenerated from the cell produced according to step    (a); and-   (c) optionally further plants are produced from the plant produced    according to step (c).

In a preferred embodiment of the method, the starch is modified in sucha way that it has an increased gel texture and/or a reduced phosphatecontent and/or a reduced peak viscosity and/or a reduced pastificationtemperature and/or a reduced size of the starch granules and/or amodified distribution of the side-chains compared to the starch ofcorresponding wild type plants.

In this context, the terms “increased gel texture”, “reduced phosphatecontent”, “reduced peak viscosity”, “reduced pastification temperature”,“reduced size of the starch granules” and “modified distribution of theside-chains” are defined as above.

As regards the genetic modification introduced according to step (a),the same applies as has been explained in a different context withregard to the plants of the invention.

The regeneration of plants according to step (b) can be achieved bymethods known to the skilled person.

Further plants according to step (b) of the method of the invention may,for instance, be produced by vegetative propagation (e.g. by means ofcuttings, tubers or through callus culture and regeneration of wholeplants) or by sexual reproduction. Preferably, the sexual reproductionis controlled, i.e. selected plants having particular properties arecross-bred and propagated.

The present invention also relates to the plants obtainable by themethod of the invention.

The present invention also relates to propagation material of plants ofthe invention as well as of the transgenic plants produced according tothe method of the invention. In this context, the term “propagationmaterial” comprises those components of the plant that are suitable forproducing progenies in a vegetative or generative way are, for example,cuttings, callus cultures, rhizomes or tubers are suitable for thevegetative propagation. Other propagation material comprises, forexample, fruit, seeds, seedlings, protoplasts, cell cultures, etc. Thepropagation material is preferred to be tubers and seeds.

Starch obtainable from the transgenic plant cells and plants of theinvention as well as from the propagation material is a further subjectmatter of the invention.

Due to the expression of a nucleic acid molecule of the invention or ofa vector of the invention, the presence of expression of which leads toan increase in the activity of a branching enzyme compared to plantcells of wild type plants that have not been genetically modified, thetransgenic plant cells and plants of the invention synthesize a starchwhich is modified with regard to its physio-chemical properties, inparticular with regard to gel texture and/or pastification behaviourand/or the size of the starch granule and/or the phosphate contentand/or the distribution of the side-chains in comparison with starchsynthesized in wild type plants.

Moreover, the present invention also relates to starches characterisedin that they have an increased gel texture and/or a reduced phosphatecontent and/or a reduced peak viscosity and/or a reduced pastificationtemperature and/or a reduced sized of the starch granules and/or amodified distribution of the side-chains.

In a particularly preferred embodiment, the present invention relates topotato starches.

In this context, the terms “increased gel texture”, “reduced phosphatecontent”, “reduced peak viscosity”, “reduced pastification temperature”,“reduced size of the starch granules” and “modified distribution of theside-chains” are defined as above.

In addition, the present invention relates to a method for producing amodified starch comprising the step of extracting the starch from aplant (cell) of the invention as described above and/or fromstarch-storing parts of such a plant. Preferably, such a method alsocomprises the step of harvesting the cultivated plants and/or thestarch-storing parts of said plants before the starch is extracted and,more preferably, also the step of cultivating plants of the inventionprior to harvesting them. The skilled person is familiar with methodsfor extracting the starch of plants or of starch-storing parts ofplants. Furthermore, methods for extracting the starch from variousstarch-storing plants have been described, e.g. in “Starch: Chemistryand Technology (ed.: Whistler, BeMiller and Paschall (1994), 2^(nd)edition, Academic Press Inc. London Ltd.; ISBN 0-12-746270-8; cf. e.g.chapter XII, page 412-468: Maize and Sorghum Starches: Production; byWatson; chapter XIII, page 469-479; Tapioca, Arrow Root and SagoStarches: Production; by Corbishley and Miller; chapter XIV, page479-490: Potato Starch: Production and Applications; by Mitch; chapterXV, page 491 to 506: Wheat Starch Production, Modification andApplications; by Knight and Oson; and chapter XVI, page 507-528: RiceStarch: Production and Applications; by Rohmer and Klem; Maize Starch:Eckhoff et al., Cereal Chem. 73 (1996), 54-57, the extraction of maizestarch on an industrial scale is usually achieved by means of theso-called wet milling)). Appliances that are usually used for methodsfor extracting starch from plant material include separators, decanters,hydrocyclones, spray dryers and fluid-bed dryers.

Starch obtainable by the method described above is also a subject matterof the present invention.

The starches of the invention can be modified according to methods knownto the person skilled in the art and are suitable for variousapplications in the foodstuff or non-foodstuff industry in an unmodifiedor modified form.

In principle, possibilities of use can be divided into two large areas.One area comprises hydrolysis products of the starch, mainly glucose andglucan building blocks obtained via enzymatic or chemical methods. Theyserve as starting material for further chemical modifications andprocesses such as fermentation. For a reduction of costs the simplicityand inexpensive carrying out of a hydrolysis method can be ofimportance. At present, the method is essentially enzymatic with use ofamyloglucosidase. It would be possible to save costs by reducing use ofenzymes. This could be achieved by changing the structure of the starch,e.g. surface enlargement of the granule, easier digestibility due to lowbranching degree or a steric structure limiting the accessibility forthe enzymes used.

The other area where starch is used as so-called native starch due toits polymeric structure can be subdivided into two further fields ofapplication:

1. Use in Foodstuffs

Starch is a classic additive for various foodstuffs, in which itessentially serves the purpose of binding aqueous additives and/orcauses an increased viscosity or an increased gel formation. Importantcharacteristic properties are flowing and sorption behaviour, swellingand pastification temperature, viscosity and thickening performance,solubility of the starch, transparency and paste structure, heat, shearand acid resistance, tendency to retrogradation, capability of filmformation, resistance to freezing/thawing, digestibility as well as thecapability of complex formation with e.g. inorganic or organic ions.

2. Use in Non-Foodstuffs

The other major field of application is the use of starch as an adjuvantin various production processes or as an additive in technical products.The major fields of application for the use of starch as an adjuvantare, first of all, the paper and cardboard industry. In this field, thestarch is mainly used for retention (holding back solids), for sizingfiller and fine particles, as solidifying substance and for dehydration.In addition, the advantageous properties of starch with regard tostiffness, hardness, sound, grip, gloss, smoothness, tear strength aswell as the surfaces are utilized.

2.1 Paper and Cardboard Industry

Within the paper production process, a differentiation can be madebetween four fields of application, namely surface, coating, mass andspraying.

The requirements on starch with regard to surface treatment areessentially a high degree of brightness, corresponding viscosity, highviscosity stability, good film formation as well as low formation ofdust. When used in coating the solid content, a corresponding viscosity,a high capability to bind as well as a high pigment affinity play animportant role. As an additive to the mass rapid, uniform, loss-freedispersion, high mechanical stability and complete retention in thepaper pulp are of importance. When using the starch in spraying,corresponding content of solids, high viscosity as well as highcapability to bind are also significant.

2.2 Adhesive Industry

A major field of application is, for instance, in the adhesive industry,where the fields of application are subdivided into four areas: the useas pure starch glue, the use in starch glues prepared with specialchemicals, the use of starch as an additive to synthetic resins andpolymer dispersions as well as the use of starches as extenders forsynthetic adhesives. 90% of all starch-based adhesives are used in theproduction of corrugated board, paper sacks and bags, compositematerials for paper and aluminum, boxes and wetting glue for envelopes,stamps, etc.

2.3 Textiles and Textile Care Products

Another possible use as adjuvant and additive is in the production oftextiles and textile care products. Within the textile industry, adifferentiation can be made between the following four fields ofapplication: the use of starch as a sizing agent, i.e. as an adjuvantfor smoothing and strengthening the burring behaviour for the protectionagainst tensile forces active in weaving as well as for the increase ofwear resistance during weaving, as an agent for textile improvementmainly after quality-deteriorating pretreatments, such as bleaching,dying, etc., as thickener in the production of dye pastes for theprevention of dye diffusion and as an additive for warping agents forsewing yarns.

2.4 Building Industry

Furthermore, starch may be used as an additive in building materials.One example is the production of gypsum plaster boards, in which thestarch mixed in the thin plaster pastifies with the water, diffuses atthe surface of the gypsum board and thus binds the cardboard to theboard. Other fields of application are admixing it to plaster andmineral fibers. In ready-mixed concrete, starch may be used for thedeceleration of the sizing process.

2.5 Ground Stabilisation

Furthermore, the starch is advantageous for the production of means forground stabilisation used for the temporary protection of groundparticles against water in artificial earth shifting. According tostate-of-the-art knowledge, combination products consisting of starchand polymer emulsions can be considered to have the same erosion- andencrustation-reducing effect as the products used so far; however, theyare considerably less expensive.

2.6 Use in Plant Protectives and Fertilizers

Another field of application is the use of starch in plant protectivesfor the modification of the specific properties of these preparations.For instance, starch is used for improving the wetting of plantprotectives and fertilizers, for the dosed release of the activeingredients, for the conversion of liquid, volatile and/or odorousactive ingredients into microcristalline, stable, deformable substances,for mixing incompatible compositions and for the prolongation of theduration of the effect due to a reduced disintegration.

2.7 Drugs, Medicine and Cosmetics Industry

Starch may also be used in the fields of drugs, medicine and in thecosmetics industry. In the pharmaceutical industry, starch may be usedas a binder for tablets or for the dilution of the binder in capsules.Furthermore, starch is suitable as disintegrant for tablets since, uponswallowing, it absorbs fluid and after a short time it swells so muchthat the active ingredient is released. For qualitative reasons, medicallubricating and vulnerary dusting powders are further fields ofapplication. In the field of cosmetics, the starch may for example beused as a carrier of powder additives, such as scents and salicylicacid. A relatively extensive field of application for the starch istoothpaste.

2.8 Starch as an Additive in Coal and Briquettes

Starch can also be used as an additive in coal and briquettes. By addingstarch, coal can be quantitatively agglomerated and/or briquetted inhigh quality, thus preventing premature disintegration of thebriquettes. Barbecue coal contains between 4 and 6% added starch,calorated coal between 0.1 and 0.5%. Furthermore, starch is suitable asa binding agent since adding it to coal and briquette can considerablyreduce the emission of toxic substances.

2.9 Processing of Ore and Coal Slurry

Furthermore, starch may be used as a flocculant in the processing of oreand coal slurry.

2.10 Additive for Casting Materials

Another field of application is the use as an additive to processmaterials in casting. For various casting processes cores produced fromsands mixed with binding agents are needed. Nowadays, the most commonlyused binding agent is bentonite mixed with modified starches, mostlyswelling starches.

The purpose of adding starch is increased flow resistance as well asimproved binding strength. Moreover, swelling starches may fulfil moreprerequisites for the production process, such as dispersability in coldwater, rehydratisability, good mixability in sand and high capability ofbinding water.

2.11 Rubber Industry

In the rubber industry starch may be used for improving the technicaland optical quality. Reasons for this are improved surface gloss, gripand appearance. For this purpose, starch is dispersed on the stickyrubberised surfaces of rubber substances before the cold vulcanization.It may also be used for improving the printability of rubber.

2.12 Production of Leather Substitutes

Another field of application for modified starch is the production ofleather substitutes.

2.13 Starch in Synthetic Polymers

In the plastics market the following fields of application are emerging:the integration of products derived from starch into the processingprocess (starch is only a filler, there is no direct bond betweensynthetic polymer and starch) or, alternatively, the integration ofproducts derived from starch into the production of polymers (starch andpolymer form a stable bond).

The use of the starch as a pure filler cannot compete with othersubstances such as talcum. This situation is different when the specificstarch properties become effective and the property profile of the endproducts is thus clearly changed. One example is the use of starchproducts in the processing of thermoplastic materials, such aspolyethylene. Thereby, starch and the synthetic polymer are combined ina ratio of 1:1 by means of coexpression to form a ‘master batch’, fromwhich various products are produced by means of common techniques usinggranulated polyethylene. The integration of starch in polyethylene filmsmay cause an increased substance permeability in hollow bodies, improvedwater vapor permeability, improved antistatic behaviour, improvedanti-block behaviour as well as improved printability with aqueous dyes.

Another possibility is the use of the starch in polyurethane foams. Dueto the adaptation of starch derivatives as well as due to theoptimisation of processing techniques, it is possible to specificallycontrol the reaction between synthetic polymers and the hydroxy groupsof the starch. The results are polyurethane films having the followingproperty profiles due to the use of starch: a reduced coefficient ofthermal expansion, decreased shrinking behaviour, improvedpressure/tension behaviour, increased water vapour permeability withouta change in water acceptance, reduced flammability and cracking density,no drop off of combustible parts, no halides and reduced aging.Disadvantages that presently still exist are reduced pressure and impactstrength.

Product development of film is not the only option. Also solid plasticsproducts, such as pots, plates and bowls can be produced by means of astarch content of more than 50%. Furthermore, the starch/polymermixtures offer the advantage that they are much easier biodegradable.

Furthermore, due to their extreme capability to bind water, starch graftpolymers have gained utmost importance. These are products having abackbone of starch and a side lattice of a synthetic monomer grafted onaccording to the principle of radical chain mechanism. The starch graftpolymers available nowadays are characterised by an improved binding andretaining capability of up to 1000 g water per g starch at a highviscosity. These super absorbers are used mainly in the hygiene field,e.g. in products such as nappies and sheets, as well as in theagricultural sector, e.g. in seed pellets.

What is decisive for the use of the novel starch modified by recombinantDNA techniques are, on the one hand, structure, water content, proteincontent, lipid content, fibre content, ashes/phosphate content,amylose/amylopectin ratio, distribution of the relative molar mass,branching degree, granule size and shape as well as crystallization, andon the other hand, the properties resulting in the following features:flow and sorption behaviour, pastification temperature, viscosity,thickening performance, solubility, paste structure, transparency, heat,shear and acid resistance, tendency to retrogradation, capability of gelformation, resistance to freezing/thawing, capability of complexformation, iodine binding, film formation, adhesive strength, enzymestability, digestibility and reactivity.

The production of modified starch by genetically operating with atransgenic plant may modify the properties of the starch obtained fromthe plant in such a way as to render further modifications by means ofchemical or physical methods superfluous. On the other hand, thestarches modified by means of recombinant DNA techniques might besubjected to further chemical modification, which will result in furtherimprovement of the quality for certain of the above-described fields ofapplication. These chemical modifications are principally known. Theseare particularly modifications by means of

heat treatment

acid treatment

formation of starch ethers

-   -   starch alkyl ether, O-allyl ether, hydroxylalkyl ether,        O-carboxylmethyl ether, N-containing starch ethers, P-containing        starch ethers and S-containing starch ethers.

formation of branched starches

formation of starch graft polymers.

oxidation and

esterification

-   -   leading to the formation of phosphate, nitrate, sulfate,        xanthate, acetate and citrate starches. Other organic acids may        also be used for the esterification.

In another embodiment, the present invention relates to parts of plantsof the invention that can be harvested, e.g. fruit, storage roots,roots, blossoms, buds, sprouts or stems, preferably seeds or tubers withsaid parts that can be harvested containing plants cells of theinvention.

In another aspect, the present invention relates to a regulatory regionwhich naturally controls, in bacterial cells, the transcription of anabove-described nucleic acid molecule of the invention encoding abranching enzyme from bacteria of the genus Neisseria.

Within the meaning of the present invention, the term “regulatoryregion” relates to a region that influences the specificity and/or theextent of the expression of a gene sequence, e.g. in such a way that theexpression takes place in response to certain external stimuli or at acertain time. Such regulatory regions usually are located in a regionthat is called promoter. Within the meaning of the present invention,the term “promoter” comprises nucleotide sequences that are necessaryfor initiating the transcription, i.e. for binding the RNA polymerase,and may also comprise the TATA box(es).

In a preferred embodiment, the regulatory region of the inventioncomprises a nucleotide sequence selected from the group consisting of:

-   (a) nucleotide sequences comprising the nucleotides 1 to 169 of the    nucleotide sequence depicted in SEQ ID NO. 1;-   (b) the nucleotide sequence of the regulatory region contained in    the insert of the plasmid DSM 12425 or parts thereof; and-   (c) nucleotide sequences hybridizing with the sequences of (a)    or (b) under stringent conditions.

The nucleotides 1 to 169 of the sequence depicted in SEQ ID NO. 1 formpart of the regulatory region of the gene of the branching enzyme fromNeisseria denitrificans. Putative promoter sequences are located at thepositions 36 to 44, 51 to 55 and 157 to 162, wherein the sequence“GGGAGA” possibly is a Shine-Dalgarno sequence.

The present invention also relates to regulatory regions having ahomology to the aforementioned regulatory regions that is so high thatthey hybridize to at least one of said regions, preferably understringent conditions. Regulatory regions that have a sequence identityof at least 80%, preferably of at least 90% and most preferably of atleast 95% to any of the aforementioned regulatory regions, in particularto the one depicted in SEQ ID NO. 1, are particularly preferred.

They also comprise regulatory regions which are modified with regard tothe above-described regulatory regions, for instance due to deletion(s),insertion(s), substitution(s), addition(s) and/or recombination(s)and/or modification(s).

The skilled person is familiar with methods for introducing suchmodifications into the regulatory regions. Moreover, the person skilledin the art knows that the regulatory regions of the invention may becoupled with further elements which influence the transcription inbacterial cells, e.g. with enhancer elements.

The present invention also relates to recombinant DNA moleculescomprising a regulatory region of the invention.

In such a recombinant DNA molecule, the regulatory region is preferredto be linked to a heterologous DNA sequence. In this context, the term“heterologous” means that said sequence is naturally not linked to theregulatory region. In addition, a recombinant DNA molecule of theinvention may contain further regulatory elements which are ofimportance as regards transcription and/or translation in bacterialcells, e.g. transcription or translation enhancers.

Moreover, the present invention relates to host cells that aretransformed with a regulatory region, a recombinant DNA molecule or avector of the invention.

Furthermore, the present invention relates to vectors containing aregulatory region of the invention or a recombinant DNA molecule of theinvention. Said vectors comprise, for instance, also plasmids, cosmids,bacteriophages, viruses, etc. which usually are used for methods inmolecular genetics.

In addition, the invention relates to an in-vitro method for producingα-1,6-branched α-1,4-glucans using the substrate sucrose and an enzymecombination of an amylosucrase and a branching enzyme. Within themeaning of the present invention, the term “in-vitro method” relates toa conversion, i.e. a reaction, which takes place outside the livingorganism. In particular, the term “in vitro” means that the method ofthe invention takes place in a reaction vessel. Most preferably, theterm “in vitro” means that the reaction takes place in absence of livingcells.

The advantage of the method of the invention is that it is possible tocontrol the branching degree and that it is possible, by means of saidcontrol, to adapt the properties of the glucans synthesized to theplanned use of the glucans. Thus, as regards the application ascapsulation material in pharmaceutics, there is the possibility ofoptimising the release rate of pharmaceutical agents by purposefullyadjusting the branching degree.

Within the meaning of the present invention, an amylosucrase(sucrose:1,4-α-D-glucan 4-α-glucosyltransferase, E.C. 2.4.1.4) is anenzyme which catalyses the conversion of sucrose to water-insolubleα-1,4-glucans and fructose. For said enzyme, the following reactionscheme is suggested:sucrose+(α-1,4-D-glucan)_(n) →D-fructose+(α-1,4-D-glucan)_(n+1)

This is a transglycosylation reaction. The products of said reaction arewater-insoluble α-1,4-glucans and fructose. The transglycosylation maytake place in the absence or in the presence of acceptor molecules. Suchacceptor molecules may be, for instance, polysaccharides likemalto-oligosaccharides, dextrin or glycogen. If said acceptor moleculeis a linear, oligomeric α-1,4-glucan, the product resulting from thetransglycosylation reaction by means of the amylosucrase is a polymericlinear α-1,4-glucan. If the transglycosylation reaction by means ofamylosucrase is carried out without any acceptor molecules, a glucanhaving a terminal fructose molecule is obtained. Within the meaning ofthe present invention, all products obtained by means of an amylosucrasein the absence or in the presence of acceptor molecules are calledα-1,4-glucans.

For the reaction mechanism of a transglycosylation by means of anamylosucrase in the absence of an acceptor molecule, the followingreaction scheme is suggested:G-F+n(G-F)→G _(n)-G-F+nF,wherein G-F is sucrose, G is glucose, F is fructose and G_(n)-G-F is anα-1,4-glucan.

For the reaction mechanism of a transglycosylation by means ofamylosucrase in the presence of an acceptor molecule, the followingreaction scheme is suggested:mG-F+G _(n) →G _(n-m) +mF,wherein G_(n) is a polysaccharide acceptor molecule, G_(n-m) is apolysaccharide consisting of an acceptor plus an α-1,4-glucan chainsynthesized thereto, G-F is sucrose, F is fructose and G is glucose.

No co-factors are necessary for the transglycosylation by means of anamylosucrase.

In principle, all amylosucrases which catalyse the synthesis of linearα-1,4-glucans starting from sucrose are suitable for carrying out themethod of the invention.

Up to now, amylosucrases from several bacteria species have been known,in particular mainly from Neisseria species (MacKenzie et al., Can. J.Microbiol. 24 (1978), 357-362).

Thus, an amylosucrase of prokaryotic origin is preferred to be used.Amylosucrases have been known, for example, from Neisseria perflava(Okada and Hehre, J. Biol. Chem. 249 (1974), 126-135; MacKenzie et al.,Can. J. Microbiol. 23 (1977), 1303-1307) or from Neisseria canis,Neisseria cinerea, Neisseria denitrificans, Neisseria sicca andNeisseria subflava (MacKenzie et al., Can. J. Microbiol. 24 (1978),357-362). Furthermore, WO 95/31553 describes an amylosucrase fromNeisseria polysaccharea. An amylosucrase that is naturally secreted by aprokaryote is preferred to be used.

In a preferred embodiment of the invention, an amylosucrase fromNeisseria polysaccharea is used.

The enzyme that is expressed in Neisseria polysaccharea is extremelystable and binds very tight to the polymerization products and iscompetitively inhibited by the reaction product fructose (MacKenzie etal., Can. J. Microbiol. 23 (1977), 1303-1307). As regards the Neisseriaspecies Neisseria polysaccharea, the amylosucrase is secreted (Riou etal., Can. J. Microbiol. 32 (1986), 909-911), whereas in other Neisseriaspecies, it remains in the cell. An amylosucrase having the amino acidsequence depicted in SEQ ID NO. 5 is particularly preferred to be used.

In another preferred embodiment of the invention, a purifiedamylosucrase is used.

In this context, a purified amylosucrase is an enzyme which issubstantially free of cellular components of the cells in which theprotein is synthesized. Preferably, the term “purified amylosucrase”relates to an amylosucrase which has a degree of purity of at least 70%,preferably of at least 85% and most preferably of at least 90%.

The use of a purified protein for producing α-1,4-glucans has variousadvantages. In contrast to methods using partially purified proteinextracts, the reaction medium of the method of the invention does notcontain any residues of the production strain (microorganism) that isused to purify the protein or to produce it by means of geneticengineering.

What is more, there are advantages in the food and pharmaceuticalindustries if the purified protein is used. The components of theproduct are defined more exactly, too, if the reaction medium is definedand if all unnecessary components have been removed. This leads to aless extensive procedure for marketing authorisation for these products,which have been manufactured by means of biotechnology, in the food andpharmaceutical industry, in particular, since said products are supposedto show no traces of a transgenic microorganism.

Within the meaning of the present invention, a branching enzyme(α-1,4-glucan:α-1,4-glucan 6-glycosyltransferase, E.C. 2.4.1.18) is aprotein catalysing a transglycosylation reaction in which theα-1,4-linkings of an α-1,4-glucan donor are hydrolyzed and the releasedα-1,4-glucan chains are transferred to an α-1,4-glucan acceptor chainand converted into α-1,6-linkings.

In principle, all branching enzymes of any origin (bacterial, fungal,plant, animal) are suitable for carrying out the method of the invention(cf. e.g. Baba et al., Biochem. Biophys. Res. Commun. 181 (1991), 87-94;Kossmann et al., Mol. Gen. Genet. 203 (1991), 237-244; Nakamura andYamanouchi, Plant Physiol. 99 (1992), 1265-1266; Baecker et al., J.Biol. Chem. 261 (1986), 8738-8743; Kiel et al., Gene (1989), 9-17,etc.).

The person skilled in the art can isolate corresponding genes by meansof standard methods of molecular biology, as have been described,amongst others, in Sambrook et al. (Sambrook et al., Molecular Cloning,A Laboratory Manual, 2^(nd) edition, Cold Spring Harbor LaboratoryPress, NY, USA (1989)).

In a preferred embodiment of the invention, the branching enzyme is abranching enzyme from a prokaryote, preferably from a bacterium of thegenus Neisseria, more preferably from Neisseria denitrificans and mostpreferably from a branching enzyme of the invention as is describedbelow. A branching enzyme having the amino acid sequence depicted in SEQID NO. 1 is particularly preferred.

In another preferred embodiment, the branching enzyme is a purifiedbranching enzyme. In this context, a purified branching enzyme is anenzyme which is substantially free of cellular components of the cellsin which the protein is synthesized. Preferably, the term “purifiedbranching enzyme” means that the enzyme has a degree of purity of atleast 70%, preferably of at least 85% and most preferably of at least90%.

Moreover, in the method of the invention, proteins are preferred to beused which have been produced recombinantly. Within the meaning of thepresent invention, said proteins are proteins which have been producedby introducing a DNA sequence encoding said protein into a host cell andexpressing it there. The protein may subsequently be recovered from thehost cell and/or the culture medium. The host cell is preferred to be abacterium or a protist (e.g. fungi, in particular yeasts, algae), suchas defined, for example in Schlegel “Allgemeine Mikrobiologie” (GeorgThieme Verlag, 1985, 1-2). In particular, the proteins are preferred tobe secreted by the host cell. Such host cells for producing arecombinant protein can be generated using methods that are known to theperson skilled in the art.

Methods in Enzymology 153 (1987), 385-516, Bitter et al. (Methods inEnzymology 153 (1987), 516-544; Sawers et al., Applied Microbiology andBiotechnology 46 (1996), 1-9; Billmann-Jacobe, Current Opinion inBiotechnology 7 (1996), 500-504; Hockney, Trends in Biotechnology 12(1994), 456-463 and Griffiths et al., Methods in Molecular Biology 75(1997), 427-440 give an overview of different expression systems.Expression vectors have been described extensively in the literature.Apart from a selection marker gene and a replication origin guaranteeingthe replication in the selected host, they usually contain a bacterialor a viral promoter, and mostly a termination signal for thetranscription. Between the promoter and the termination signal, there isat least one restriction site or a polylinker which allow the insertionof an encoding DNA sequence. The DNA sequence which naturally controlsthe transcription of the corresponding gene can be used as promotersequence if it is active in the selected host organism. Said sequence,however, may also be exchanged for other promoter sequences. Bothpromoters effecting the constitutive expression of the gene andinducible promoters allowing a directed regulation of the expression ofthe downstream gene can be used. Bacterial and viral promoter sequenceshaving these properties have been described extensively in theliterature. Regulatory sequences for the expression in microorganisms(e.g. E. coli, S. cerevisiae) have been described sufficiently in theliterature. Promoters allowing a particularly strong expression of thedownstream gene include, for example, the T7 promoter (Studier et al.,Methods in Enzymology 185 (1990), 60-89), lacuv5, trp, trp-lacUV5(DeBoer et al., in Rodriguez and Chamberlin (eds.) Promoters, Structureand Function; Praeger, N.Y. (1982), 462-481; DeBoer et al., Proc. Natl.Acad. Sci. USA (1983), 21-25),

p1, rac (Boros et al., Gene 42 (1986), 97-100). Normally, the amounts ofproteins reach their top level from the middle to about the end of thelogarithmic phase of the growth cycle of the microorganisms. Therefore,preferably inducible promoters are used for the synthesis of proteins.These inducible promoters often result in a higher yield of proteinsthan the constitutive promoters. Due to the constant transcription andtranslation of a cloned gene, the use of strong constitutive promotersoften has the effect that the energy for other essential cell functionsis lost and that, thus, the cell growth is slowed down (Bernard R.Glick/Jack J. Pasternak, Molekulare Biotechnologie (1995), SpektrumAkademischer Verlag GmbH, Heidelberg Berlin Oxford, p. 342). Hence, atwo-step method is often used to achieve the optimum amount of proteins.First, the host cells are cultivated under optimum conditions until theyreach a relatively high cell density. In the second step, thetranscription is induced depending on the kind of promoter used. In thiscontext, a tac promoter that is inducible by lactose or IPTG(=isopropyl-β-D-thiogalacto-pyranoside) is particularly suitable (DeBoeret al., Proc. Natl. Acad. Sci. USA 80 (1983), 21-25). Terminationsignals for the transcription have also been described in theliterature.

The transformation of the host cell with the DNA encoding acorresponding protein DNA can normally be carried out according tostandard methods, as described, for instance, in Sambrook et al.(Molecular Cloning: A Laboratory Course Manual, 2^(nd) edition (1989),Cold Spring Harbor Press, New York). The host cell is cultivated inculture media which correspond to the needs of the respective host cell.In particular, pH value, temperature, salt concentration, aeration,antibiotics, vitamins and trace elements, etc. are taken intoconsideration.

The enzyme produced by the host cells can be purified according tostandard purification techniques, such as precipitation, ion exchangechromatography, affinity chromatography, gel filtration, HPLC reversephase chromatography, etc.

By modifying the DNA expressed in the host cells, it is possible toproduce a polypeptide in the host cell, which is easier to be isolatedfrom the culture medium due to certain properties. Thus, there is thepossibility of expressing the protein to be expressed as a fusionprotein together with another polypeptide sequence the specific bindingproperty of which allows the isolation of the fusion protein throughaffinity chromatography (e.g. Hopp et al., Bio/Technology 6 (1988),1204-1210; Sassenfeld, Trends Biotechnol. 8 (1990), 88-93).

In a preferred embodiment of the method of the invention, enzymes areused which have been produced recombinantly and which have been secretedby the host cell into the culture medium so that it is not necessary todisrupt cells or to purify the protein any further since the secretedprotein may be recovered from the supernatant. Methods known in processengineering, such as dialysis, reverse osmosis, chromatographic methods,etc. may be used for removing residual components of the culture medium.The same applies to the reconcentration of the protein secreted into theculture medium. Normally, the secretion of proteins by microorganisms ismediated by N-terminal signal peptides (signal sequence, leaderpeptide). Proteins having said signal sequence may pass through the cellmembrane of the microorganism. Secretion of proteins may be achieved bylinking the DNA sequence that encodes said signal peptide to thecorresponding region encoding the enzyme.

A signal peptide that optionally occurs naturally is preferred, e.g. thesignal peptide of the amylosucrase from Neisseria polysaccharea.

The signal peptide of the α-CGTase from Klebsiella oxytoca M5A1 (Fiedleret al., J. Mol. Biol. 256 (1996), 279-291) or a signal peptide as isencoded by the nucleotides 11529-11618 of the sequence accessible in theGenBank under the accession number X86014 is particularly preferred.

As an alternative, the enzymes used in the method of the invention mayalso have been produced using an in-vitro transcription and translationsystem which leads to the expression of the proteins without usingmicroorganisms.

In another preferred embodiment, the amylosucrase and/or the branchingenzyme are immobilized on a support material.

Immobilizing the enzymes has the advantage that the enzymes can berecovered from the reaction mixture in a simple manner as catalysts ofthe synthesis reaction and can be used several times. Since thepurification of enzymes usually requires much time and money,immobilization and recycling can save costs considerably. The degree ofpurity of the reaction products which do not contain any remainingproteins is another advantage.

There is a plurality of support materials at disposal for immobilizingproteins wherein the coupling with the support material may take placevia covalent or non-covalent bindings (for an overview see: Methods inEnzymology 135, 136, 137). For example, agarose, alginate, cellulose,polyacrylamide, silica or nylon are extensively used as supportmaterial.

In another preferred embodiment of the method, a (partially purified)enzyme crude extract of an amylosucrase and/or a branching enzyme isused. In this context, a crude extract is an amylosucrase and/orbranching enzyme preparation having a reduced degree of purity incomparison with a purified enzyme (cf. Examples 5 and 6).

In a preferred embodiment, in the method of the invention the branchingdegree of the α1,6-branched α-1,4-glucans is modified by changing theratio of the protein activity of branching enzyme and amylosucrase. Inthis context, the ratio of the protein activity is the ratio of theprotein activities (u) from amylosucrase and branching enzyme. Theprotein activities may be determined as described in Examples 7 and 8.When the method of the invention is carried out (cf. Example 9), theratio of protein activity (units of amylosucrase/units of branchingenzyme) may range from 1/4000 to 2000/1.

In a preferred embodiment, the ratio of the protein activity ranges from1/1500 to 1500/1.

In another preferred embodiment, the ratio of the protein activityranges from 1/800 to 1300/1.

In a particularly preferred embodiment, the ratio of the proteinactivity ranges from 1/400 to 1200/1.

It is possible to modify the branching degree of the α-1,6-branchedα-1,4-glucans obtained from 0.05% to 35% by changing the ratio of theprotein activity.

In a preferred embodiment, it is possible to change the branching degreeof the α-1,6-branched α-1,4-glucans in 6-position from 0.15% to 25%,more preferably from 0.20% to 15% and most preferably from 0.25% to 12%.

If the method of the invention is used, it is possible, in particular,to produce products having a higher branching degree than glycogen.

Within the meaning of the present invention, the branching degree is theaverage share of branchings in O-6 position compared to all glucoseunits linked differently. The branching degree can be determined bymethylation analysis (cf. Example 10).

In another preferred embodiment, in the method of the invention, themolecular weight of the products is modified by changing the proteinactivity ratio. It is, in particular, possible to change the proteinactivity ratio during the reaction that leads to the synthesis of theα-1,6-branched α-1,4-glucans.

In another preferred embodiment of the method of the invention, themethod is to be carried out at different sucrose concentrations. Inprinciple, it is possible for the method to be carried out at aconcentration preferably ranging from 1% to 80% sucrose (w/v), morepreferably ranging from 5% to 50% and most preferably from 10% to 40%.

In the present invention, the molecular weight is determined by lightscattering experiments (Light Scattering from Polymer Solutions, editor:Huglin, M. B., Academic Press, London, 1972) according to Berry (J.Chem. Phys. 44 (1966), pp. 4550). By means of the method of theinvention, it is possible, in particular, to adjust the molecular weightof the α-1,6-branched α-1,4-glucans produced by said method to a rangeof 1000 to 3000×10₆. Preferably, the α-1,6-branched α-1,4-glucans have amolecular weight ranging from 100,000 to 1500×10₆, more preferably from100,000 to 1000×10₆, even more preferably from 262,000 to 1000×10₆ andmost preferably from 262,000 to 499×10₆.

Furthermore, the invention relates to α-1,6-branched α-1,4-glucansobtainable by the above-described method of the invention. Saidα-1,6-branched α-1,4-glucans have a branching degree which is higherthan the one that is achieved if only the activity of an amylosucrase isused and which is 25 mol % at the most.

In a preferred embodiment of the invention, these are α-1,6-branchedα-1,4-glucans having a branching degree ranging from 0.05% to 20%,preferably from 0.15% to 17%, more preferably from 0.2% to 15%, evenmore preferably from 0.25% to 13% and most preferably from 0.3% to 12%.In another preferred embodiment of the invention, the branching degreeranges from 0.35% to 11% and, in particular, from 0.4% to 10.5%.

The α-1,6-branched α-1,4-glucans of the invention can be used in thefood and non-food industries as has been described above with regard tothe starch of the invention.

The plasmid pBB48, which has been produced within the present invention,was deposited with the Deutsche Sammlung von Mikroorganismen andZellkulturen (DSMZ, German Collection of microorganisms and cellcultures) in Braunschweig, which is approved as internationaldepository, on 25 Sep. 1998 with the accession number DSM 12425according to the requirements of the Budapest Treaty.

FIG. 1 schematically shows the structure of the plasmid pBB48 (DSM12425).

FIG. 2 shows a number of α-1,4-glucans having a varying degree ofα-1,6-branchings which were produced by means of the method of theinvention and which were subsequently dyed with Lugol's solution.

From left to right: amylosucrase (left), amylosucrase+decreasing amountsof branching enzyme activity. The maximum absorption of thecorresponding samples were: 615 nm, 483 nm, 500 nm, 526 nm, 534 nm, 560nm, 577 nm.

FIG. 3 shows a HPLC chromatograph of a highly branched process product(A) which has been debranched with isoamylase and a rat liver glycogensample (B) which has been debranched with isoamylase.

FIG. 4 shows the scheme of the methylation analysis.

FIG. 5 shows a diagram of the results of the analysis of sample 7described in Examples 9 and 10 after one and after two methylationsteps. The values for the 2,3,6-methylation are 96.12% and 96.36%,respectively.

FIG. 6 shows a graphic illustration of the shares in terminal (“2346Me”) and 6-linked (“23 Me”) glucose units of the glucan samplesexamined.

FIGS. 7 and 8 show gas chromatographs of the samples 3 and 7 describedin the Examples.

FIG. 9 schematically shows the plasmid pBE-fnr-Km.

FIG. 10 shows an activity gel for the branching enzyme.

FIG. 11 shows the schematic illustration of an RVA profile.

FIG. 12 shows the distribution of granule size of the lines 143-13A and143-59A compared to the wild type.

FIG. 13 shows the microscopic magnification of the starch granules ofthe lines 143-13A, 143-34A and 143-59A in comparison with the starchgranules of wild type plants (light microscope by Leitz, Germany).

FIG. 14 shows the gel texture of the starches of different transgeniclines compared to starches from wild type plants. The texture wasdetermined by means of a texture analyzer.

FIG. 15 shows the RVA profile of the starches of the lines 143-11A,143-13A, 143-59A compared to the wild type.

FIGS. 16 to 18 show the results of HPLC chromatographies which representthe pattern of the distribution of the side-chains of the lines 143-WT(=wild type), 143-13A and 143-59A.

FIG. 19 shows the elution gradient that was used for thechromatographies depicted in FIGS. 16 to 18.

FIG. 20 shows the percentage deviation of side-chains having certainchain lengths of the starches analysed in FIGS. 16 to 18 from the wildtype.

The following Examples illustrate the invention.

Materials:

-   disruption buffer: 100 mM Tris/HCl, pH 8.5; 5 mM Na₂EDTA; 2 mM DTT;    1 mM Pefabloc®-   washing buffer: 50 mM Tris/HCl, pH 8.5; 5 mM Na₂EDTA; 10% glycerol-   HIC buffer: 50 mM potassium phosphate buffer, pH 7.0; 5 mM EDTA; 2    mM DTT; 10% glycerol-   oyster glycogen type II from oyster (Sigma G8751)    Methods:    Starch Analysis    (a) Determination of the Amylose/Amylopectin Ratio

Starch was isolated from potato plants according to standard methods andthe ratio of amylose to amylopectin was determined according to themethod described by Hovenkamp-Hermelink et al. (Potato Research 31(1988), 241-246).

(b) Determination of the Phosphate Content

In starch, the positions C2, C3 and C6 of the glucose units may bephosphorylated.

For determining the content of phosphate groups at the C6 position, 100mg starch was hydrolysed in 1 ml 0.7 M HCl for 4 hours at 95° C.(Nielsen et al., Plant Physiol. 105 (1994), 11-117). After neutralisingwith 0.7 M KOH, 50 ml of the hydrolysate were subjected to anoptical-enzymatic test for determining the glucose-6-phosphate. At 334nm, the change in the absorption of the test mixture (100 mMimidazole/HCl; 10 mM MgCl₂; 0.4 mM NAD; 2 unitsglucose-6-phosphate-dehydrogenase from Leuconostoc mesenteroides; 30°C.) was determined.

The overall content of phosphate was determined according to the methodby Ames (Methods in Enzymology VIII (1966), 115-118).

Approximately 50 mg starch are added to 30 μl of an ethanolic magnesiumnitrate solution and ashed for 3 hours at 500° C. in a muffle furnace.300 μl 0.5 M hydrochloric acid were added to the residue and incubatedfor 30 min at 60° C. Then, an aliquot is filled up to 300 μl 0.5 Mhydrochloric acid, added to a mixture of 100 μl of 10% ascorbic acid and600 μl of 0.42% ammonium molybdate in 2 M sulphuric acid and incubatedfor 20 min at 45° C.

Then, a photometric determination at 820 nm is carried out with acalibration curve using phosphate standards.

(c) Determination of the Gel Texture (Texture Analyzer)

2 g starch (TS) are pasted in 25 ml H₂O (cf. RVA) and subsequentlysealed airtight and stored at 25° C. for 24 hours. The samples are fixedunder the probe (round stamp) of a texture analyzer TA-XT2 by StableMicro Systems and the gel texture is determined with regard to thefollowing parameters:

test speed 0.5 mm/s penetration depth 7 mm contact area 113 mm2 pressure2 g(d) Viscosity Profile

2 g starch (TS) are added to 25 ml H₂O and put in a Rapid Visco Analyzer(Newport Scientific Pty Ltd., Investment Support Group, Warriewod NSW2102, Australia) for analysis. The device was operated according to themanufacturer's instructions. For determining the viscosity of theaqueous solution of the starch, first of all, the starch suspension isheated from 50° C. to 95° C. at a speed of 12° C. per minute. Then, thetemperature is maintained for 2.5 minutes at 95° C. Subsequently, thesolution is cooled down from 95° C. to 50° C. at a speed of 12° C. perminute. The viscosity is determined during the whole time.

The pastification temperature is determined by means of the slope of theviscosity graph depending on the time. If the slope of the graph ishigher than 1.2 (this value is set by the user), the computer programidentifies the temperature measured in this moment as pastificationtemperature.

(e) Determination of Glucose, Fructose and Sucrose

The content of glucose, fructose and sucrose is determined according tothe method described by Stitt et al. (Methods in Enzymology 174 (1989),518-552).

(f) Analysis of the Distribution of the Side-Chains of the Amylopectin

The distribution of the side-chains and the preparation are determinedas described in Lloyd et al. (Biochem. J. 338 (1999), 515-521). It ispointed to the fact that, using said method, only the amylopectin isdebranched and that the amylose is separated from the amylopectin beforedebranching by means of thymol precipitation. The following conditionsfor the elution are selected (simplified illustration, the exact elutionprofile is shown in FIG. 19):

time 1M NaAc min 0.15M NaOH % in 0.15M NaOH % 0 100 0 5 100 0 20 85 1535 70 30 45 68 32 60 0 100 70 0 100 72 100 0 80 100 0(g) Determination of the Granule Size

The size of the granules was determined with a photosedimentometer ofthe type “Lumosed” by Retsch GmbH, Germany.

The distribution of the granule size was determined in an aqueoussolution and was carried out according to the manufacturer's indicationsas well as on the basis of the literature, e.g. H. Pitsch,Korngröβenbestimmung; LABO-1988/3 Fachzeitschrift für Labortechnik,Darmstadt.

(h) Determination of the Water-Binding Capacity

For determining the water-binding capacity, the residue was weighedafter separating the soluble parts of the starch swelled at 70° C. bymeans of centrifugation. The water-binding capacity (WBV) of the starchwas determined with reference to the initial weight that was correctedby the soluble mass.WBV (g/g)=(residue−(initial weight−soluble proportion))/(initialweight−soluble proportion).

EXAMPLE 1 Isolation of a Genomic DNA Sequence Encoding a BranchingEnzyme from Neisseria denitrificans

For isolating the branching enzyme from Neisseria denitrificans, firstof all, a genomic library was established. For this purpose, cells ofNeisseria denitrificans of the strain deposited as ATCC 14686 at theATCC were cultivated on Columbia blood agar plates and subsequentlyharvested. The genomic DNA was isolated and purified according to themethod by Ausubel et al. (in: Current Protocols in Molecular Biology(1987); J. Wiley & Sons, NY). After a partial restriction digestion withthe restriction endonuclease Sau3A, a ligation with BamHI-cleaved phagevector DNA (lambdaZAPExpress by Stratagene) was carried out. After thein-vivo excision of the phage library, the plasmids obtained weretransformed into the E. coli mutant (PGM-) (Adhya and Schwartz, J.Bacteriol. 108 (1971), 621-626). When growing on maltose, said mutantforms linear polysaccharides which turn blue after colouring withiodine. 60,000 transformants were plated onto YT agar plates with IPTG(1 mM), kanamycin (12.5 mg/l) and maltose (1%) and after incubation for16 hours at 37° C., they were vaporized with iodine. 60 bacteriacolonies which had a red, brown or yellow colour after vaporization withiodine were sleeted and plasmid DNA was isolated therefrom(Birnboim-Doly, Nucleic Acid Res. 7, 1513-1523). The isolated plasmidswere then used for retransformation of the same E. coli-(PGM)-mutant(Adhya and Schwartz, J. Bacteriol. 108 (1971), 621-626). After repeatedplating and vaporization with iodine, the clones could be reduced from60 isolates to 4 isolates. A restriction analysis was carried out withthese four plasmids showing an EcoRI fragment (1.6 kb) which had thesame size in all four plasmids (FIG. 1).

EXAMPLE 2 Sequence Analysis of the Genomic Fragment of the Plasmid pBB48

The 1.6 kb EcoRI fragment was isolated (Geneclean, Bio101) from a cloneobtained according to Example 1 (pBB48) which had an approx. 3.9 kbinsert in the vector pBK-CMV (Stratagene). For DNA sequencing, thefragment was cloned into the vector pBluescript which had been cleavedwith EcoRI. The plasmid obtained in this way was sequenced. Then, theentire DNA sequence encoding the branching enzyme as well as thesequence of flanking regions was determined by means of the startingplasmid pBB48 (SEQ ID NO. 1). The plasmid pBB48 is shown in FIG. 1. Theplasmid is deposited under DSM 12425.

EXAMPLE 3 Expression of the Branching Enzyme in Recombinant E. coliCells

In general, an endogenous branching enzyme (glgB) is expressed in the E.coli laboratory strains. For this reason, the G6MD2 mutant of E. coliwas used for detecting the branching enzyme activity. The strain E. coliHfr G6MD2 (E. coli Genetic Stock Center, Yale University, CGSC#5080) hasan extended deletion in the region of the glucan synthesis genes (glgA,glgB, glgC). For detecting the branching enzyme activity, said mutantwas transformed with the plasmid pBB48 and a crude extract was preparedof the propagated cells. The proteins of said crude extract wereseparated electrophoretically in a polyacrylamide gel and then incubatedwith and without rabbit phosphorylase B (100 mM sodium citrate, pH 7.0;AMP, glucose-1-phosphate) for determining the branching enzyme activity.Violet bands only appeared in the gel stimulated with phosphorylase,which indicated a strong branching enzyme activity.

EXAMPLE 4 In-Vitro Production of α-1,6-Branched α-1,4-Glucans withProtein Crude Extracts in a Cell-Free System

For the expression of the branching enzyme, the mutant E. coli G6MD2 wastransformed with the plasmid pBB48. The cells were cultivated with YTmedium with kanamycin (12.5 mg/l) for 16 hours while shaking in anErlenmeyer flask. After centrifugation (5000×g), the pellet obtained waswashed with 100 mM Tris/HCl, pH 7.5, 1 mM DTT and, after suspension inthe same buffer, the cells were disrupted with an ultrasonic probe. Byanother centrifugation (10,000×g), the cell debris was separated fromthe soluble proteins and a yellowish supernatant having a proteinconcentration of approx. 10 mg/ml was obtained.

From the protein crude extract obtained in that manner, differentamounts (100 μl, 10 μl, 1 μl, 0.1 μl, 0.01 μl, 0.001 μl) were added toan unchanged amount of an amylosucrase in 50 ml 100 mM sodium citrate,pH 7.0 with 20% sucrose and 0.02% sodium azide. After a few hours, afirst clouding was observed in the reaction mixture. After three days,the mixture was centrifuged and the products formed were washed withdeionized water.

The products are soluble in DMSO and may be characterised by measuringan absorption spectrum with Lugol's solution by means of which thebranching degree of the products formed may be estimated. For thispurpose, the DMSO solution was strongly diluted with water and Lugol'ssolution was added and the spectrum from 400 nm to 700 nm wasimmediately measured in a Beckmann spectrophotometer (cf. FIG. 2).

Separation of the side-chains that were split off with isoamylase on aCarbopak PA100 column by means of HPLC (DIONEX; running agent: 150 mMNaOH with 1 M sodium acetate gradient) shows the same pattern for astrongly branched product as for a rat liver glycogen debranched withisoamylase (FIG. 3).

After incubation with a pullulanase, the side-chains were only split offto a very small extent.

EXAMPLE 5 Purification of the Branching Enzyme and N-Terminal Sequencingof the Protein

For isolating the branching enzyme of Neisseria denitrificans fromrecombinant Hfr G6MD2 E. coli cells (see above), which had beentransformed with pBB48, first an overnight culture of said cells wascentrifuged. The cell precipitate was then suspended in 3 volumesdisruption buffer and disrupted in the French press at a pressure ofapprox. 16,000 to 17,000 psi. After centrifugation at 10,000 g for onehour, the supernatant was diluted to reach the 4-fold volume by addingwashing buffer. Then, it was bound to DEAE cellulose DE52 using thebatch-method and filled into a chromatography column which was washedwith 2 to 3 column volumes of washing buffer. Subsequently, a linear 1 MNaCl gradient was applied for elution. The fractions with branchingenzyme activity were combined (see Example 8), (NH₄)₂SO₄ was added(final concentration 20% (w/v)) and applied to a TSK butyl Toyopearl650M column. After washing with 2 to 3 column volumes of HIC buffer, towhich additionally an ammonium sulphate solution with a degree ofsaturation of 20% (114 g ammonium sulphate per liter) had been addedbefore, the branching enzyme was eluted in HIC buffer using an ammoniumsulphate gradient that falls linearly from 20% to 0%. Fractions withbranching enzyme activity were combined. For concentrating the protein,the purification step with the combined fractions was subsequentlyrepeated using a small TSK butyl Toyopearl 650M column (Tose Haas(Montgomery Ville, Pa.)). The purified protein was then applied to apolyacrylamide gel, blotted onto a PVDF membrane, dissolved again andsequenced N-terminally by WITA GmbH, Teltow, Germany, according to theEdman method. The sequence obtained was: MNRNXH (SEQ ID NO. 3).

EXAMPLE 6 Purification of an Amylosucrase

For producing an amylosucrase, E. coli cells were used which had beentransformed with a DNA encoding an amylosucrase from Neisseriapolysaccharea. The DNA has the nucleotide sequence depicted in SEQ IDNO. 4 and is derived from a genomic library of N. polysaccharea.

An overnight culture of said E. coli cells which secrete theamylosucrase from Neisseria polysaccharea was centrifuged off andresuspended in approx. 1/20 volume of 50 mM sodium citrate buffer (pH6.5), 10 mM DTT (dithiothreitol), 1 mM PMSF(phenylmethylsulfonylfluoride). Then, the cells were disrupted twicewith a French press at 16,000 psi. Subsequently, 1 mM MgCl₂ andbenzonase (by Merck; 100,000 units; 250 units μl⁻¹) were added to thecell extract in a final concentration of 12.5 units ml⁻¹. After that,the mixture was incubated at 37° C. for at least 30 min while shakinggently. The extract was left to stand on ice for at least 1.5 hours.Then, it was centrifuged at 4° C. for 30 min at approx. 40,000 g untilthe supernatant was relatively clear.

A pre-filtration with a PVDF membrane (Millipore “Durapore”, or similar)was carried out which had a pore diameter of 0.45 μm. The extract wasleft to stand over night at 4° C. Before carrying out theHI-(hydrophobic interaction) chromatography, solid NaCl was added to theextract and adjusted to a concentration of 2 M NaCl. Then, the mixturewas again centrifuged at 4° C. for 30 min at approx. 40,000 mg.Subsequently, the remaining residues of E. coli were removed from theextract by filtering it with a PVDF membrane (Millipore “Durapore” ofsimilar) which had a pore diameter of 0.22 μm. The filtered extract wasseparated on a butylsepharose-4B column (Pharmacia) (volume of thecolumn: 93 ml, length: 17.5 cm). Approx. 50 ml of the extract having anamylose activity of 1 to 5 units μl⁻¹ were applied to the column. Then,non-binding proteins were washed off the column with 150 ml buffer B(buffer B: 50 mM sodium citrate, pH 6.5, 2 M NaCl). Finally, theamylosucrase was eluted by means of a falling linear NaCl gradient (from2 M to 0 M NaCl in 50 mM sodium citrate in a volume of 433 ml at aninflux rate of 1.5 ml min⁻¹) which had been generated by means of anautomatic pumping system (FPLC, Pharmacia). The elution of theamylosucrase occurred between 0.7 M and 0.1 M NaCl. The fractions werecollected, desalted on a PD10 sephadex column (Pharmacia), stabilisedwith 8.7% glycerol, examined for amylose sucrose activity and finallydeep-frozen in storage buffer (8.7% glycerol, 50 mM citrate).

EXAMPLE 7 Determination of the Amylosucrase Activity

The amylosucrase activity was determined by incubating purified proteinor protein crude extract in different dilutions at 37° C. in 1 mlreaction mixtures containing 5% sucrose, 0.1% dextrin and 100 mMcitrate, pH 6.5. After 0 min, 30 min, 60 min, 120 min, 180 min, 240 min,300 min and 360 min, 10 μl each are taken from said mixture, and theenzymatic activity of the amylosucrase is stopped by immediate heatingto 95° C. Then, the proportion of the fructose released by theamylosucrase is determined in a combined photometric test. 1 μl to 10 μlof the inactivated sample are put in 1 ml 50 mM imidazole buffer, pH6.9, 2 mM MgCl₂, 1 mM ATP, 0.4 mM NAD₊ and 0.5 U/ml hexokinase. Aftersequential addition of glucose-6-phosphate dehydrogenase (fromLeuconostoc mesenteroides) and phosphoglucose isomerase, the change inthe absorption is measured at 340 nm. Subsequently, the amount offructose released is calculated by means of the Lambert-Beer law.

If the value obtained is brought into relation with the time when thesample is taken, the number of units (1 U=μmol fructose/min) (per μlprotein extract or μg purified protein) can be determined.

EXAMPLE 8 Determination of the Enzyme Activity of a Branching Enzymefrom Neisseria denitrificans

The enzymatic activity of the branching enzyme was determined inaccordance with a method described in the literature (Krisman et al.,Analytical Biochemistry 147 (1985), 491-496; Brown and Brown, Meth.Enzymol. 8 (1966), 395-403). The method is based on the principle ofreduced iodine binding-affinity of branched glucans in comparison withnon-branched α-1,4-glucans.

For determining the enzymatic activity of the branching enzyme, a seriesof samples of various dilutions of the branching enzyme was put into acooled micro-titre plate. Then, the reaction was started by adding 190μl of an amylose reaction mixture (preparation see below) and incubatedat 37° C. in an incubator. Exactly after 30 min, the reaction wasstopped by adding 100 μl of Lugol's solution (0.5 mM) and the sampleswere measured in a micro-titre reading device (Molecular Devices) at 650nm. A mixture without amylose served as control. The reference samplewith the maximum extinction value which contained amylose but nobranching enzyme had an OD₆₅₀ of 1.2.

In order to be able to better compare independent assays, only thesample dilution is used for the calculation which leads to a decrease ofthe OD₆₅₀ by 0.5 units during an incubation time of 30 min

Definition of an Activity Unit (U) of the Branching Enzyme:

The amount of enzymes causing a decrease of the OD₆₅₀ by 0.5 units from1.2 to 0.7 in 30 min in the test described is half a unit of thebranching enzyme.

Preparation of the Amylose Reaction Mixture:

While stirring, 1 ml of a 0.5% amylose solution (manufacturer: Fluka;amylose from potato) w/v in DMSO are added to 10 ml sodium citratebuffer (100 mM, pH 6.5, 0.02% w/v NaN₃). For measuring, the clear stocksolution is again diluted with sodium citrate buffer to a ratio of 1:4to 1:8. In the test, absorption with Lugol's solution should be at 1.2in the reference sample used (maximum).

EXAMPLE 9 Production of α-1,6-Branched α-1,4-Glucans Having DifferentBranching Degrees

For producing α-1,6-branched α-1,4 glucans having different branchingdegrees, purified amylosucrase from Neisseria polysaccharea (cf. Example6) and a purified branching enzyme from Neisseria denitrificans (cf.Example 5) were added to a 20% sucrose solution (w/v) in a reactionvolume of 10.86 ml. Depending on the test mixture, the two enzymes wereused in different protein activity ratios to each other (for thedetermination amylosucrase see Example 7; for the determination of thebranching enzyme see Example 8) (see Table 1):

amylosucrase preparation: 6.2 U/mg; 1.8 mg/ml

branching enzyme preparation: 75 U/mg; 6.9 mg/ml

TABLE 1 units Amsu/ no. μl BE μl Amsu units BE units Amsu units BE 1 725140 375 1.6    1/234.4 2 181.3 140 94 1.6   1/58.8 3 45.5 140 24 1.6  1/15 4 11.4 140 5.90 1.6   1/3.7 5 2.8 140 1.45 1.6 1.1/1 6 0.713 1400.37 1.6 4.3/1 7 0.179 140 0.09263 1.6 17.3/1  8 0.0446 140 0.02308 1.669.3/1  9 0.0112 140 0.00580 1.6 275.9/1  10 0.0028 140 0.00145 1.61103.4/1   11 0 140 0 1.6 — 13 glycogen from Mytillus edulis — BE =branching enzyme Amsu = amylosucrase units = for determination seeExamples 7 and 8

EXAMPLE 10 Determination of the Branching Degree by Means of MethylationAnalysis

The branching degree of the glucans obtained was subsequently determinedby means of a methylation analysis.

1. Examinations Carried Out

-   -   methylation of all free OH-groups of the glucan samples, each        time double determination    -   hydrolysis of the permethylated polymers followed by a reduction        at C-1 and acetylation of the monomer mixture    -   gas chromatographic analysis and quantification of the reaction        products

The branching degree of the glucan samples was established by means of amethylation analysis (cf. FIG. 4). The free OH-groups of the polymer arelabelled by conversion into methylether.

The degradation to monomers is carried out in an acid hydrolytic mannerand leads to partially methylated glucose molecules which are present inpyranosidic/furanosidic form and as α- and α-glucosides. These variantsare focussed by reduction with NaBH₄ in the corresponding partiallymethylated sorbite derivative. By subsequent acetylation of freeOH-groups the reaction products can be examined by means of gaschromatography.

The following table shows the texture and the DMSO solubility of theglucans obtained.

TABLE 2 DMSO solubility DMSO solubility sample Texture (cold) (100° C.)1 plastic foam-like (+) + (slightly cloudy colourless solution) 2 n.d.n.d. n.d. 3 plastic foam-like (+) + (slightly cloudy colourlesssolution) 4 n.d. n.d. n.d. 5 colourless powder + + 6 n.d. n.d. n.d. 7colourless powder + + 8 n.d. n.d. n.d. 9 colourless powder + + 10 n.d.n.d. n.d. 11 colourless powder + + 13 yellowish powder (+) + n.d. = notdetermined2. Experimental Part

a) Preparation of the DMSO Solutions

1% solutions (w/v) were prepared in DMSO. Not all of the samples werewell-soluble at room temperature: 1, 3 and 13 had to be heated for 30minutes to 110° C. Apart from the solutions 1 and 3, which were slightlycloudy, there were optically clear solutions (cf. Table 2).

b) Methylation

2 ml of the DMSO solution (i.e. 20 mg polymer) were transferred to a 50ml-nitrogen flask, added to 5 equivalents/OH (eq/OH) of freshly prepareddimsyl solution in an N2 stream and stirred for 30 minutes. Thesolutions turned cloudy and viscous. The content of the flask was frozenin an ice-bath, 10 eq/OH methyliodide were added and, after thawing, themixture was stirred for at least 2 hours. Before the seconddeprotonation and methylation step, surplus methyliodide was removed inthe vacuum.

After removing the surplus methyliodide, processing was carried out byadding 50 ml water and after extracting 5 times with 10 mldichloromethane each. Any traces of DMSO were removed from the organicphase by extracting 3 times with water, then the organic phase was driedwith CaCl₂, filtered and concentrated. The products were clear,yellowish films.

By means of sample 7, it was first checked how many methylation stepsare necessary for the permethylation of the hydroxyl groups. After thefirst methylation, half of the mixture was processed, the other half wasmethylated again. After both samples had been degraded, the results ofthe GC-analyses were compared. First, it was found that the reaction hadalmost been quantitatively after one methylation step (cf. FIG. 5). Foridentifying a possible branching at C-3, which also may only seem to bepresent due to submethylation at said position, a second methylation wascarried out in any case.

FIG. 5 shows a diagram of the results of the analysis of sample 7 afterone and after two methylation steps; the values for 2,3,6-methylationare 96.12% and 96.36%, respectively.

c) Hydrolysis

2 mg of the methylated sample were weighed-in in a 1 ml-pressure glass,0.9 ml 2 M trifluor acetic acid were added and it was stirred for 2.5hours at 120° C. After cooling the glass, the mixture was concentratedin an Na stream. For removing traces of acid, three times toluene wasadded and blown off.

TABLE 3 Data of the methylation sample 1 sample 3 sample 5 sample 7sample 9 sample 11 sample 13 method 1 initial weight 21.9 22.7 21.7 32.523.4 22.6 23.5 (mg) (mmol) 0.135 0.140 0.134  0.200 0.144 0.139 0.145resulting weight 30.4 29.2 28.0 25₁₎ 27.7 28.8 30.4 (mg) (mmol) 0.1490.143 0.137  0.122₁₎ 0.136 0.141 0.149 % of theory 110 102 102 —₁₎ 94101 103 method 2 initial weight 23.7 22.1 20.7 20.8 23.1 21.5 19.5 (mg)(mmol) 0.146 0.136 0.128  0.128 0.142 0.133 0.120 resulting weight 31.130.6 27.5 16.0₂₎ 31.4 29.4 25.5 (mg) (mmol) 0.152 0.150 0.135  0.078₂₎0.154 0.144 0.125 % of theory 104 110 105 61₂₎ 108 108 104 ₁₎Half ofthis sample was already taken and processed after the first methylationstep, thus, no exact data was available. ₂₎The small amount is due to anerror in processing.

d) Reduction

0.5 ml of an 0.5 M ammoniacal NaBD4 solution was added to the remainderof the previous reaction step and stirred for 1 hour at 60° C. Thereagent was carefully destroyed with a few drops of glacial acetic acid.The resulting borate was removed by adding five times a 15% methanolicacetic acid and subsequently blowing off as boric acid trimethylester.

e) Acetylation

50 μl pyridine and 250 μl acetic acid anhydride was added to theremainder of the previous reaction step and stirred for 2 hours at 95°C. After cooling, the reacting mixture was dripped into 10 ml saturatedNaHCO₃ solution and extracted five times with dichloromethane. Thereaction products in the organic phase were examined by means of gaschromatography (product, cf. FIG. 4).

f) Gas Chromatography

The examinations by means of gas chromatography were carried out using adevice by Carlo Erby GC 6000 Vega Series 2 with on-column inlet and FIDdetector. The separations were conducted on a fused-silica capillarycolumn called Supelco SPB5 (inner diameter 0.2 mm, length 30 m) usinghydrogen as carrier gas and a pressure of 80 kPa. The followingtemperature programme was used: 60° C. (1 min) −25° C./min→130° C. −4°C./min→280° C.

3. Results

The gas chromatographs were analysed by identifying the peaks,integrating the peak areas and correcting the data by means of the ECRconcept by Sweet et al. (Sweet et al., Carbohydr. Res. 40 (1975), 217).

The 1,6-anhydro-compounds that could be observed in samples 1 and 3 aredue to the high branching degree at C-6. During hydrolysis, this leadsto monomers having a free OH-group at C-6 which may further react toform these derivatives under the reaction conditions. When calculatingthe branching degree, these proportions have to be added to the “2,3-Me”value.

FIG. 6 is an illustration of the proportions of terminal (“2346Me”) and6-linked (“23”Me) glucose units of the glucan samples examined.

TABLE 4 Results of the analysis in mol %: the abbreviations (A, B, etc.)correspond to the ones in FIG. 1; “16AnhPy” = 1,6-anhydro-4-O-acetyl-2,3-di-O-methyl-D-glucopyranose, “16AnhFu” = 1,6anhydro-5-O-acetyl-2,3-di-O-methyl-D-glucofuranose; “Me1” and “Me2”denote two independent methylation analyses of the respective samples.sample 1 sample 3 average average Me1 Me2 value Me1 Me2 value 16AnhPy0.37 traces 0.19 traces traces — 16AnhFu 0.53 0.47 0.50 traces traces —2346-Me (A) 11.73 11.94 11.84 9.49 10.68 10.08 234-Me (B) traces traces— — — — 236-Me (C) 76.37 77.80 77.09 82.97 80.67 81.82 23-Me (D) 9.759.16 9.46 7.54 8.34 7.94 26-Me (E) 0.45 0.31 0.38 traces 0.32 0.16 36-Me0.44 0.31 0.38 traces traces — 2-Me 0.20 — 0.10 — — — 3-Me — — — — — —6-Me — — — — — — Un-Me 0.20 — 0.10 — — — sample 5 sample 7 averageaverage Me1 Me2 value Me1 Me2 value 16AnhPy — — — — — — 16AnhFu — — — —— — 2346-Me (A) 2.42 2.51 2.47 2.60 2.77 2.69 234-Me (B) — — — — — —236-Me (C) 95.54 96.18 95.86 96.36 96.89 96.63 23-Me (D) 1.36 1.05 1.210.48 0.33 0.41 26-Me (E) 0.37 traces 0.19 0.26 traces 0.13 36-Me 0.300.26 0.28 0.29 traces 0.15 2-Me — — — — 3-Me — — — — 6-Me — — — — Un-Me— — — — sample 9 sample 11 average average Me1 Me2 value Me1 Me2 value16AnhPy — — — — — — 16AnhFu — — — — — — 2346-Me (A) 2.89 2.79 2.84 2.602.49 2.55 234-Me (B) — — — — — — 236-Me (C) 95.62 95.62 95.62 96.2197.20 96.70 23-Me (D) 0.67 0.69 0.68 0.52 0.31 0.42 26-Me (E) 0.36 0.420.39 0.36 traces 0.18 36-Me 0.47 0.48 1.47 0.30 traces 0.15 2-Me — — — —— — 3-Me — — — — — — 6-Me — — — — — — Un-Me — — — — — — sample 13 Me1Me2 average value 16AnhPy traces traces — 16AnhFu traces traces —2346-Me (A) 8.91 7.46 8.19 234-Me (B) traces traces — 236-Me (C) 83.7185.45 84.58 23-Me (D) 7.07 6.87 6.97 26-Me (E) 0.32 0.22 0.27 36-Metraces traces — 2-Me — — — 3-Me — — — 6-Me — — — Un-Me — — —

EXAMPLE 11 Production of α-1,6-Branched α-1,4-Glucans Having DifferentMolecular Weights

For producing α-1,6-branched α-1,4-glucans having different molecularweights, a purified amylosucrase from Neisseria polysaccharea (cf.Example 6) and a purified branching enzyme from Neisseria denitrificans(cf. Example 5) were added to a 20% sucrose solution (w/v) in a reactionvolume of 10.86 ml. Depending on the test mixture, the two enzymes wereused in different protein activity ratios (for the determination of theamylosucrase activity see Example 7; for the branching enzyme seeExample 8) (cf. Table 1). The molecular weights and the radius ofinertness R_(g) were determined by means of light scattering (LightScattering from Polymer Solutions; editor: Huglin, M. B., AcademicPress, London, 1972). The dried samples 1-11 were dissolved in DMSO, H₂O(at a ratio of 90:10) and different dilutions (approx. 2.5 g/l to 0.25g/l) were analysed in a device for measuring the light scattering(SOFICA, Societé française d'instruments de contrôle et d'analyses. LeMesnil Saint-Denis, France). The data obtained in this way were [ . . .]¹ according to Berry (J. Chem. Phys. 44 (1966), 4550 et seq.). ¹translator's note: verb missing.

TABLE 5 ratio of radius of amylosucrase:branching inertness molecularweight sample enzyme Rg in nm in g/mol 1 0.05 104 282 × 10₆ 2 0.2 154499 × 10₆ 3 0.8 76 228 × 10₆ 4 3.21 64  76 × 10₆ 5 12.84 63  20 × 10₆ 651.22 38  1.1 × 10₅ 7 204.03 277 472,000 8 818.87 n.d. n.d. 9 3275.49170 469,000 10 13043.48 n.d. n.d. 11 no branching enzyme 143 262,000 13glycogen 14.3 1.59 × 10₆ g/mol (sea mussels) (Burchard, W.:Macromolecules 10: 919 (1977)) n.d. = not determined

EXAMPLE 12 Construction of an Expression Cassette for TransformingPlants for the Plastidial Expression of a Branching Enzyme fromNeisseria denitrificans

The oligonucleotides BE-5′ and BE-3′ (SEQ ID NO. 6 and SEQ ID NO. 7)were used for amplifying the sequence coding for the branching enzymefrom Neisseria denitrificans by means of PCR starting from the plasmidpBB48 (deposited with the Deutsche Sammlung von Mikroorganismen andZellkulturen (DSMZ, German Collection of microorganisms and cellcultures) in Braunschweig with the accession number DSM 12425). Theresulting amplified sequences therefrom were digested with therestriction endonucleases SalI and SdaI and cloned into the plasmidpBinAR-fnr which was cleaved with SalI and SdaI. The plasmid resultingtherefrom was denoted pBE-fnr-Km (FIG. 9).

Conditions for the PCR:

Buffer and Polymerase by Boehringer Mannheim (Pwo Polymerase No.:1644947)

DNA 0.2 ng 10 × buffer + MgSO₄ 5 μl dNTPs (10 mM each) 1 μl primer BE-5′120 nM primer BE-3′ 120 nM Pwo polymerase 1.0 units distilled water ad50 μlReaction Conditions

step 1 95° C. 2:00 min step 2 95° C. 0:30 min step 3 66° C. 0:30 minstep 4 72° C. 2:00 min (plus 1 sec. per cycle) step 5 72° C. 8:00 minSteps 2 to 4 were repeated in 40 cycles.

The plasmid pBE-fnr-Km was used for transforming potato plants accordingto standard methods (see above).

EXAMPLE 13 Identification and Detection of Transgenic Potato Plants withBranching Enzyme Activity

By means of Northern blot analysis, it was possible to identify from thetransgenic potato plants produced according to Example 12 plants whichdisplayed an mRNA of a branching enzyme from Neisseria denitrificans.For detecting the activity of the branching enzyme in the stablytransformed plants, leaf material of the plants to be examined wasdeep-frozen in liquid nitrogen and then ground in a mortar pre-cooledwith liquid nitrogen. Before the ground material thawed, extractionbuffer was added (50 mM sodium citrate, pH 6.5, 4 mM DTT, 2 mM calciumchloride). Approx. 200 μl extraction buffer were added to approx. 100 mg(fresh weight) of plant material. Solid components of the suspension ofground plant material and extraction buffer were separated by means ofcentrifugation (10,000×g). An aliquot of the clear supernatant obtainedtherefrom was mixed with a quarter of the extraction volume of runningbuffer (40% glycerol, 250 mM Tris, pH 8.8, 0.02% bromophenol blue) andseparated in polyacrylamide gel (see below) at a constant intensity ofcurrent of 20 mA per gel. (Before the protein extracts were applied, anelectrophoresis of the gels was carried out for 20 min under theconditions indicated above). After the dye bromophenol blue in therunning buffer had run out of the gel, the electrophoresis was stopped.Then, the gel was equilibrated five times in washing buffer (100 mMsodium citrate, pH 6.5) at room temperature at a volume that was fivetimes the gel volume for 20 minutes each while stirring. Subsequently,the gel was incubated in incubation buffer (100 mM sodium citrate, pH6.5, 5% sucrose, 0.625 units of purified amylosucrase from Neisseriapolysaccharea (for purification of the enzyme and determination of theactivity see above)) in an amount that is five times the amount of thegel volume at 30° C. for 16 hours. After decanting the incubation bufferand after adding Lugol's solution (diluted at a ratio of 1:5), theglucan which is formed by the amylosucrase in combination with thebranching enzyme becomes visible as bluish-brown band (FIG. 10). Theentire remaining polyacrylamide gel turns blue due to the amylosucraseactivity in the incubation buffer.

Composition of the Polyacrylamide Gel:

a) Separation Gel

375 mM Tris, pH 8.8

7.5% polyacrylamide (Biorad no. EC-890)

For the Polymerization:

1/2000 volumes TEMED

1/100 volumes ammonium persulfate

b) Collection Gel

125 mM Tris, pH 6.8

4% polyacrylamide (Biorad no. EC-890)

For the Polymerization:

1/2000 volumes TEMED

1/100 volumes ammonium persulfate

c) Electrophoresis Buffer

375 mM Tris, pH 8.8

200 mM glycine

EXAMPLE 14 Analysis of the Starch of Plants Having an IncreasedBranching Enzyme Activity

According to standard techniques, starch was isolated from transgenicpotato plants which had been produced according to Examples 12 and 13and examined with regard to its physical and chemical properties. It wasfound that the starch formed by the transgenic potato plants differsfrom starch synthesized in wild type plants, for example in itsphosphate content and in the viscosity and pastification propertiesdetermined by means of RVA. The results of the physico-chemicalcharacterisation of the modified starches based on the above-describedanalysis techniques are shown in the following table.

phos- phate amyl- gel in ose RVA RVA RVA RVA RVA tex- C6 con- max. min.fin. set. T ture no. genotype (%) tent (%) (%) (%) (%) (%) (%) 1 Desiree100 22.0 100 100 100 100 100 100 (wild type) 2 143-13A 36 20.9 50 83 8279 79 162 3 143-11A — 22.5 92 90 88 80 99.5 — 4 143-59A 22 20.9 36 69 78114 99 225 legend: 143-13A, 143-11A, 143-59A = transgenic potato plantswhich over-express the branching enzyme from Neisseria denitrificans.RVA = Rapid Visco Analyzer. max = maximum viscosity = peak viscosity min= minimum viscosity fin. = viscosity at the end of the measurement set.= setback = difference between min. and fin. T = pastificationtemperature Except for the amylose content, the percentage values referto the wild type (=100%).

The results of the RVA analysis, the analysis of the distribution of thesize of the starch granules and the gel texture are also shown in FIGS.11 to 15.

Furthermore, FIGS. 16 to 18 show the results of the HPLCchromatographies which illustrate the pattern of the distribution of theside-chains of the lines 143-WT (=wild type), 143-13A and 143-59A. FIG.19 shows the elution gradient used in connection with the HPLC analysis.In FIG. 20, the percentage deviation of side-chains having a certainchain length from the wild type is shown.

The following two tables explain how the proportions of side-chains werecalculated.

TABLE 7 143-59A (measurement 1) 143-59A (measurement 2) proportionproportion average value name of the sum of the sum of the area of thepeak area [%] peak area [%] proportions peak A 2 B 2 C 2 D 2 E 2 DP 6577122 4.5 690167 5.08 4.79 DP 7 504371 3.93 544770 4.01 3.97 DP 8341520 2.66 377170 2.77 2.72 DP 9 387706 3.02 462686 3.40 3.21 DP 10511664 3.99 602911 4.43 4.21 DP 11 684394 5.34 776228 5.71 5.52 DP 12884346 6.90 976001 7.18 7.04 DP 13 1038389 8.10 1138027 8.37 8.23 DP 141080589 8.43 1175544 8.65 8.54 DP 15 1046585 8.16 1144404 8.42 8.29 DP16 977127 7.62 1016555 7.48 7.55 DP 17 850092 6.63 881777 6.49 6.56 DP18 720854 5.62 739080 5.44 5.53 DP 19 626277 4.88 627135 4.61 4.75 DP 20526159 4.10 522122 3.84 3.97 DP 21 439356 3.43 431106 3.17 3.30 DP 22354956 2.77 336907 2.48 2.62 DP 23 281320 2.19 266412 1.96 2.08 DP 24224165 1.75 200219 1.47 1.61 DP 25 176641 1.38 169596 1.25 1.31 DP 26152651 1.19 145821 1.07 1.13 DP 27 153046 1.19 123171 0.91 1.05 DP 28117125 0.91 103599 0.76 0.84 DP 29 92294 0.72 85067 0.63 0.67 DP 3073885 0.58 59729 0.44 0.51 Σ A 2 Σ C 2 sum 12822634 100.00 13596204100.00 100.00

The peak areas in columns A 1, A 2, C 1 and C 2 have been determined bymeans of the application program AI 450, version 3.31 by Dionex.

TABLE 8 143-WT (measurement 1) 143-WT (measurement 2) proportionproportion average value name of the sum of the sum of the area of thepeak area [%] peak area [%] proportions peak A 2 B 2 C 2 D 2 E 2 DP 6123190 1.75 160046 1.68 1.72 DP 7 95526 1.36 137396 1.45 1.40 DP 8 873651.24 126639 1.33 1.29 DP 9 158742 2.26 210845 2.22 2.24 DP 10 3085444.39 382957 4.03 4.21 DP 11 465107 6.61 581774 6.12 6.36 DP 12 5748828.17 721814 7.59 7.88 DP 13 634154 9.01 796824 8.38 8.70 DP 14 6335669.01 798684 8.40 8.70 DP 15 594327 8.45 766484 8.06 8.25 DP 16 5375377.64 699141 7.35 7.50 DP 17 470522 6.69 609229 6.41 6.55 DP 18 4030815.73 539584 5.67 5.70 DP 19 352504 5.01 486633 5.12 5.06 DP 20 3137084.46 432720 4.55 4.51 DP 21 265289 3.77 385358 4.05 3.91 DP 22 2117223.01 323248 3.40 3.20 DP 23 179015 2.54 274938 2.89 2.72 DP 24 1487582.11 227219 2.39 2.25 DP 25 119135 1.69 197839 2.08 1.89 DP 26 1039021.48 177493 1.87 1.67 DP 27 88686 1.26 147919 1.56 1.41 DP 28 67024 0.95131325 1.38 1.17 DP 29 61086 0.87 104515 1.10 0.98 DP 30 37850 0.5487704 0.92 0.73 Σ A 1 Σ C 1 sum 7035222 100.00 9508328 100.00 100.00

The invention claimed is:
 1. Starch prepared by the process thatcomprises extracting starch from transgenic plants or parts thereof thatcomprise transgenic plant cell containing a nucleic acid molecule linkedto regulatory sequences guaranteeing the transcription in plant cells,wherein said nucleic acid molecule encodes a branching enzyme from abacterium of the genus Neisseria selected from the group consisting of(a) nucleic acid molecules encoding a protein which comprises the aminoacid sequence depicted in SEQ ID NO. 2; (b) nucleic acid moleculescomprising the coding region depicted in SEQ ID NO. 1; (c) nucleic acidmolecules encoding a protein which comprises the amino acid sequenceencoded by the insert in plasmid DSM 12425; (d) nucleic acid moleculescomprising the coding region for a branching enzyme, which is containedin the insert of the plasmid DSM 12425; and (e) nucleic acid moleculesencoding a protein the sequence of which has, in the first 100 aminoacids, a homology of at least 65% to the amino acid sequence depicted inSEQ ID NO. 2; wherein said starch has a phosphate content that is atlast 20% reduced as compared to starch from a corresponding wild typeplant.
 2. The starch according to claim 1, wherein the composition ofthe starch is modified in such a way that it has at least one of theproperties selected from the group consisting of an increased geltexture, a reduced phosphate content, a reduced peak viscosity, areduced pastification temperature, a reduced size of starch granules anda modified distribution of side-chains when compared to the starch fromcorresponding wild type plant.
 3. The starch according to claim 1,wherein said starch has a modified distribution of side-chains in thatthe proportion of side-chains with a DP of 6 to 8 is increased by atleast 25% in comparison to the proportion of side-chains with thecorresponding degree of polymerization of amylopectin from acorresponding wild-type plant.
 4. The starch according to claim 1,wherein said protein sequence has a homology of at least 95% to theamino acid sequence depicted in SEQ ID NO. 2.